Molecules associating to c-terminal domain in receptor cell

ABSTRACT

Concerning intracellular signal transduction mechanism, there has been drawn a novel hypothesis that, even in the case where phosphorylation does not occur in the intracellular C-terminal domain of a receptor, an unknown molecule associates with the Pro-C terminal domain of a G protein-coupled receptor for each chemokine and thus leukocyte chemotaxis depending on the receptor is controlled. To examine this hypothesis and clarify therapeutic targets in inflammatory diseases as well as other various diseases, attempts are made to search for a CCR2-binding protein. As a result, a novel cytoplasmic protein associating directly and specifically with the Pro-12-C-terminal domain of CCR2 is found out and it is clarified that this protein forms clusters with CCR2 after stimulation with CCL2. Thus, it is confirmed that there is a novel signal transduction system in the G protein relating signal transduction in the CCL2-CCR2 pathway. It is also found out that this novel protein associates with the intracellular C-terminal domain of a receptor CCR5 too.

TECHNICAL FIELD

This invention relates to a protein which associates with theintracellular C-terminal domain of a G protein-coupled receptor forchemokine, a DNA encoding the same, and utilization thereof. Morespecifically, it relates to a novel protein which associates with theintracellular C-terminal domain of receptors CCR2 and CCR5 to therebycontrol the functions of the receptors CCR2 and CCR5, a DNA encoding thesame and utilization thereof in the field of medicine.

BACKGROUND ART

In cells, external stimulation with a ligand is transmitted into cellsvia a receptor. In the case of chemokine receptors, for example, it isknown that leukocyte chemotaxis, which is the fundamental function ininflammation and immune responses, is strictly controlled by attractorsserving as agonists (Documents 1 to 4). More specifically speaking, achemokine prototype CCL2 (also known as MCAF and MCP-1) was found as amacrophage attractant mediated by a receptor CCR2 (Documents 5 to 8).Moreover, chemokines CCL3, 4 and 5 are known as agonists for CCR5.

It has been considered that intracellular signal transduction of areceptor depends on the G protein switching mechanism. Concerningchemokines, there have been identified more than 50 types of chemokinesand 20 types of G protein-coupled receptors (GPCRs) as thechemokine-chemokine receptor family. Each chemokine receptor has astrict chemokine-specificity and shows an expression pattern restrictedto leukocyte subtype (Documents 9 to 11).

Now, the relationships between the intracellular signal transduction ofreceptors and diseases will be discussed. In the case of the chemokinereceptor CCR2, for example, the receptor CCR2 is a 7-transmembrane Gprotein-coupled receptor known as occurring in monocytes, macrophages,lymphocytes, endothelial cells, smooth muscular cells and so on and itsagonist CCL2 acts as an attractant via interaction with the receptorCCR2 (Document 12). It is considered that the CCL2-CCR2 pathwayparticipates in the causes of atherosclerosis (Documents 13 and 14),chronic glomerulonephritis (Document 15), multiple sclerosis (Documents16 and 17) and other chronic inflammatory diseases (Documents 18 to 21).On the other hand, it is reported that CCR5, which is known as achemokine CCL2 (MIP-1α), CCL4 (MIP-1β) or CCL5 (RANTES) receptor, isexpressed in monocytes and macrophages and participates in variousinflammatory diseases similar to CCR2. Furthermore, it is known thatCCR2 and CCR5 serve as coreceptors for cell entry of humanimmunodeficiency virus (HIV) (Deng et al., Nature, 381, 661-666(1996)).However, a large number of points still remain unknown in intracellularsignal transduction cascades relating to leukocyte chemotaxis.Accordingly, there is a great worth in studying the mechanismcontrolling chemokine receptor-mediated leukocyte chemotaxis.

It is considered that a receptor having an attractant bonded theretoactivates a G protein and thus causes the dissociation of the G proteininto α- and β-subunits and the formation of a second messenger, therebyinitiating actin polymerization and leukocyte chemotaxis (Documents 22and 23). The dissociation of the G protein is followed by thephosphorylation of the receptor by protein kinases such as PKs, Jaks andGRKs (Documents 24 and 25). Subsequently, the phosphorylation in theintracellular domain in the carboxyl terminal region (C-terminal region)of the receptor promotes receptor internalization with adaptors Aps andarrestin and inhibits excessive responses (Document 26). As the resultsof conventional studies, there have been known GPCR-binding moleculesregulating receptors' functions such as cell-surface transportation andintracellular uptake (Documents 33 and 34). However, there have beenknown so far few GPCR-binding molecules controlling intracellularchemotactic signal cascades specific to individual receptors.

Studies on mutations have clarified that receptors binding toattaractants such as cAMP, fMLP and chemokines activate chemotacticsignal cascades even in the case where phosphorylation does not occur inthe intracellular C-terminal domain (Documents 27 to 32). In theprevious studies, GPCR-binding molecules, which regulate receptors'functions such as cell-surface transportation and intracellular uptake,were identified (Documents 33 and 34). However, chemotactic signalcascades of individual receptors are scarcely known.

It is reported that the second cytoplasmic loop and the C-terminaldomain of a GPCR are important sites in the activation of thechemotactic signal cascade by the binding to a G protein and activationthereof (Documents 27, 28, 35 and 36). When 12 residues in theintracellular C-terminal domain of CCR2 (a sequence in the Pro-12-Cterminal domain; SVFFRKHITKRF (SEQ ID NO:41)) is removed, for example,its chemotactic response disappears though the G protein-binding abilityto CCL2 remains unchanged (Documents 28 and 37). When a shorter sequencefrom the terminus is removed, however, no effect is observed. It isinteresting that the DRY motif at the second cytoplasmic loop of achemokine receptor is completely conserved while the sequence in theneighborhood of the terminus is scarcely conserved. Although chemokinereceptors CCR2 and CXCR4 both activate monocyte chemotaxis cascades, thePro-12-C terminal domain of CCR2 never relates to the conservation of asimilar domain of CXCR4.

DISCLOSURE OF THE INVENTION

Based on a series of facts as discussed above, the present inventorsdrawn a novel hypothesis concerning the intracellular signaltransduction mechanism that, even in the case where phosphorylation doesnot occur in the intracellular C-terminal domain of a receptor, anunknown molecule associates with the Pro-C terminal domain of a Gprotein-coupled receptor for each chemokine and thus controls leukocytechemotaxis depending on the receptor. To examine this hypothesis andclarify therapeutic targets in inflammatory diseases as well as othervarious diseases, they attempted to search for a CCR2-binding protein asan aspect of the invention. As a result, they found out a novelcytoplasmic protein associating directly and specifically with thePro-12-C-terminal domain of CCR2 and clarified that that this proteinforms clusters with CCR2 after stimulation with CCL2. Thus, it wasconfirmed that there is a novel signal transduction system in the Gprotein relating signal transduction in the CCL2-CCR2 pathway. It wasalso found out that this novel protein associates with the intracellularC-terminal domain of a receptor CCR5 too.

Based on the above-described findings, the present inventors attemptedto compare and discuss the conservation states of corresponding geneticinformation in biological systems and clarify the mechanisms ofintracellular signal transduction and regulation concerning cellmigration, thereby developing a novel route for the establishment oftechnical means contributing to the treatment and diagnosis of diseasesin which the CCL2-CCR2 pathway and the CCL3, 4 or 5-CCR5 pathwayparticipate.

Accordingly, the present invention relates to the following DNAs.

(1) A DNA encoding the amino acid sequence of a polypeptide (a FROUNTprotein) having an amino acid sequence represented by any of SEQ IDNOS:1 to 18.

(2) A DNA encoding a FROUNT protein represented by any of SEQ ID NOS:19to 36.

(3) A DNA of the sequence having at least 90% identity to the DNA asdescribed in the above (1) or (2) and encoding a polypeptide having afunction of the FROUNT protein.

The present invention further relates to the following protein orpolypeptide.

(4) A FROUNT protein having an amino acid sequence represented by any ofSEQ ID NOS:1 to 18.

(5) A polypeptide having an amino acid sequence having at least 90%identity to the amino acid sequence as described in the above (4) andhaving the function of the FROUNT protein.

The present invention further relates to the following antisenses orribozyme.

(6) An antisense DNA or an antisense RNA inhibiting the expression of aFROUNT protein having an amino acid sequence represented by any of SEQID NOS:1 to 18.

(7) An antisense DNA or an antisense RNA directed against the fulllength or a part of the DNA as described in any of the above (1) to (3).

(8) An antisense RNA having the full length or a part of the sequencerepresented by SEQ ID NO:39.

(9) An antisense RNA having at least 90% identity to the sequence of theRNA as described in the above (8) and inhibiting the expression of aFROUNT protein.

(10) A DNA for producing the RNA as described in the above (8) or (9)which consists of the DNA sequence represented by SEQ ID NO:40 or thefull length or a part of a sequence having at least 90% identity to thissequence.

(11) A ribozyme against an RNA corresponding to a DNA encoding the aminoacid sequence represented by SEQ ID NO: 1 or the DNA sequencerepresented by SEQ ID NO:19.

The present invention further relates to the following plasmids orliposome preparations.

(12) A plasmid containing the DNA as described in any of the above (1)to (3).

(13) A liposome preparation containing the DNA as described in any ofthe above (1) to (3).

(14) A plasmid containing the DNA as described in the above (7) or (10).

(15) A liposome preparation containing the DNA or RNA as described inany of the above (7) to (10).

(16) A liposome preparation containing the ribozyme as described in theabove (11).

(17) An isolated antibody binding specifically to the polypeptide asdescribed in the above (4) or (5).

The present invention further relates to the following pharmaceuticalcompositions and treating compositions.

(18) A composition for treating chronic inflammatory disease orautoimmune diseases or for treating or preventing infectious diseaseswhich contains as the active ingredient the plasmid or the liposomepreparation as described in any of the above (14) to (16).

(19) A composition for treating atherosclerosis, chronicglomerulonephritis or multiple sclerosis, an immunomodulator or anantiallergic agent which contains as the active ingredient the plasmidor the liposome preparation as described in any of the above (14) to(16).

(20) A pharmaceutical composition which contains as the activeingredient the DNA as described in any of the above (1) to (3).

(21) An immunoenhancer, a self-defensive reaction promoter or acomposition for treating infectious diseases which contains as theactive ingredient the DNA as described in any of the above (1) to (3).

(22) An immunoenhancer, a self-defensive reaction promoter or acomposition for treating infectious diseases which contains as theactive ingredient the plasmid or the liposome preparation as describedin the above (12) or (13).

The present invention further relates to the following examinationmethod and probe to be used therein.

(23) A method of examining the presence or absence of an abnormality inthe CCL2-CCR2 pathway or the CCL3, 4 or 5-CCR5 pathway characterized bycomprising comparing the full length or a part of the DNA sequence asdescribed in any of the above (1) to (3) with a DNA sequence collectedfrom a specimen and thus judging whether or not the DNA collected fromthe specimen has an abnormality.

(24) A probe for examining the presence or absence of an abnormality inthe CCL2-CCR2 pathway or the CCL3, 4 or 5-CCR5 pathway which consists ofthe full length or a part of a sequence complementary to the DNA asdescribed in any of the above (1) to (3).

The present invention further relates to the following methods ofidentifying an inhibitor and substances to be used therein.

(25) A method of identifying an agonist inhibitor characterized bycomprising preparing a cell in which a receptor undergoingclusterization by stimulation with an agonist and a marker-labeledmolecule coupling or associating with the intracellular terminus of thereceptor are forcibly expressed, treating the cell with a specimencontaining the agonist and a candidate for the agonist inhibitor,observing whether or not the clusterization of the marker is induced inthe cell, and thus judging whether or not the candidate has aninhibitory effect on the agonist.

(26) A method of identifying an inhibitor of an agonist to receptor(s)CCR2 and/or CCR5 characterized by comprising forcibly expressing amarker-labeled FROUNT protein in a cell having the receptor(s) CCR2and/or CCR5 or expressing the same, treating the cell with an agonist toCCR2 and/or CCR5 and a candidate for the agonist inhibitor, observingwhether or not the clusterization of the receptor(s) is induced, andthus judging whether or not the candidate has an inhibitory effect onthe agonist.

(27) A method of identifying an agonist inhibitor by using a chimericreceptor cell characterized by comprising preparing a cell having alabeled FROUNT protein and a chimeric receptor by forcibly expressing achimeric receptor, which is obtained by integrating a DNA sequenceencoding the full length or a part of a FROUNT protein-associationsequence in the intracellular C-terminal domain of receptor(s) CCR2and/or CCR5 into the intracellular C-terminal domain of the DNA sequenceof a desired receptor, in a cell appropriate for the desired receptorand then forcibly expressing a marker-labeled FROUNT protein in thecell, treating the chimeric receptor cell with an agonist to thereceptor and a candidate for an agonist inhibitor, then observingwhether or not the clusterization of the receptor is induced and thusjudging whether or not the candidate has an inhibitory effect on theagonist.

(28) The identification method as described in the above (27)characterized in that the FROUNT protein-association sequence in theintracellular C-terminal domain of receptor(s) CCR2 and/or CCR5 is theamino acid sequence represented by SEQ ID NO:41.

(29) The identification method as described in the above (26) or (27)characterized in that the marker-labeled FROUNT protein is a FROUNTprotein fused with a visible color fluorescent protein.

(30) The identification method as described in the above (29) whereinthe visible color fluorescent protein is a green fluorescent protein, ared fluorescent protein, a blue fluorescent protein or a yellowfluorescent protein.

(31) A DNA encoding the FROUNT protein fused with a visible colorfluorescent protein as described in the above (29) or (30).

(32) A plasmid containing the DNA sequence as described in the above(31).

(33) A chimeric receptor DNA obtained by integrating a DNA sequenceencoding the full length or a part of a FROUNT protein-associationsequence in the intracellular C-terminal domain of receptor CCR2 intothe intracellular C-terminal domain of the DNA sequence of a desiredreceptor.

(34) The chimeric receptor as described in the above (32) characterizedin that the FROUNT protein-association sequence in the intracellularC-terminal domain of receptor(s) CCR2 and/or CCR5 is the amino acidsequence represented by SEQ ID NO:41.

(35) A cell wherein a receptor undergoing clusterization by stimulationwith an agonist and a marker-labeled molecule coupling or associatingwith an intracellular terminus of the receptor are forcibly expressed.

(36) A cell wherein a marker-labeled FROUNT protein is forciblyexpressed and receptor(s) CCR2 and/or CCR5 are further expressedtherein.

(37) A cell having a labeled FROUNT protein and a chimeric receptorprepared by forcibly expressing a chimeric receptor, which is obtainedby integrating a DNA sequence encoding the full length or a part of aFROUNT protein-association sequence in the intracellular C-terminaldomain of receptor(s) CCR2 and/or CCR5 into the intracellular C-terminaldomain of the DNA sequence of a desired receptor, in a cell appropriatefor the desired receptor and then forcibly expressing a marker-labeledFROUNT protein in the cell.

(38) The cell as described in the above (36) or (37) characterized inthat the FROUNT protein-association sequence in the intracellularC-terminal domain of receptor(s) CCR2 and/or CCR5 is the amino acidsequence represented by SEQ ID NO:41.

(39) The cell as described in the above (36) or (37) characterized inthat the marker-labeled FROUNT protein is a FROUNT protein fused with avisible color fluorescent protein.

(40) The cell as described in the above (39) wherein the visible colorfluorescent protein is a green fluorescent protein, a red fluorescentprotein, a blue fluorescent protein or a yellow fluorescent protein.

(41) A method of identifying an intracellular signal transductionpathway inhibitor depending on binding of a FROUNT protein to a receptorwhich comprises using the binding activity of the FROUNT protein to thereceptor as an indication and screening a substance inhibiting thebinding activity.

(42) The method of identifying an inhibitor as described in the above(25), (26) or (27) characterized in that the identification is madedepending on a color change as an indication by using a cell whereinboth of the receptor and the protein associating with the C-terminaldomain of the receptor are labeled with visible color markers beingdifferent from each other in color.

(43) A cell wherein both of a receptor and a protein associating withthe C-terminal domain of the receptor, which are labeled with visiblecolor markers being different from each other in color, are expressedtherein.

(44) A method of judging whether or not a specimen contains a cytotoxicsubstance which comprises treating the cell as described in the above(35) to (40) or (43) with the specimen, then treating it with an agonistto the receptor carried by the cell and observing whether or notclusterization or colocalization is induced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a test for confirming an interaction betweenthe FROUNT protein obtained from the clone 19 and the receptor CCR2using Y190 cells.

FIG. 2 shows the results of a test for confirming an interaction betweenthe FROUNT protein obtained from the clone 19 and the receptor CCR2using the coimmnoprecipitation method.

FIG. 3 shows the relationship between the intracellular C-terminaldomain sequence of CCR2 and the action of FROUNT protein 1.

FIG. 4 shows the amino acid sequence structure of human FROUNT protein1.

FIG. 5(a) shows the Kyte-Doolittle hydropathy plot of FROUNT protein 1.FIG. 5(b) shows the structural analysis of human FROUNT protein 1through database motif searching and a comparison among organisms. FIG.5(c) is a schematic model showing human FROUNT protein domains.

FIG. 6 presents fluorescent microphotographs of control eGFP (upper) andFROUNT fused with a fluorescent protein (lower) FIGS. 6(a) and 6(b)respectively show micrographs before and after treating with CCR2antibody.

FIG. 7 shows the structure of clone 1 (656 amino acids (a.a.)).

FIG. 8 shows the structure of clone 2 (611 a.a.).

FIG. 9 shows the structure of clone 13 (630 a.a.).

FIG. 10 shows the structure of clone 14 (566 a.a.).

FIG. 11 shows the structure of clone 17 (518 a.a.).

FIG. 12 shows the structure of a human FROUNT vector.

FIG. 13 shows the results of the quantification of human FROUNT mRNA andprotein in each established cell line.

FIG. 14 presents confocal microscopic images of human FROUNT protein ineach established cell line.

FIG. 15 shows the results of the detection of chemotactic activity.

FIG. 16 shows the results of the measurement of the ability of calciuminflux.

FIG. 17 shows the results of the detection of the ability ofclusterization. FIG. 17(a) presents images of stimulation of each cellswith CCR2-specific antibody, while FIG. 17(b) shows the results of theconfocal microscopic quantification of the clusterization ability ofeach receptor stimulated with the CCR2- or CXCL2-specific antibody (thelongitudinal axis referring to control %).

FIG. 18 is a schematic model of a retrovirus vector pEGFPMY which is amouse FROUNT protein vector.

FIG. 19 shows the results of a chemotaxis experiment in mice stimulatedwith thioglycolate.

FIG. 20 shows inhibition of chemokine receptor clusterization byMCP-1-specific antibody. FIG. 20(a) indicates a control case, while FIG.20(b) shows the inhibition results.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides an intracellular signaltransduction-regulating polypeptide associating with the intracellularC-terminal domain of a receptor CCR2, which is one of G protein-coupledreceptors for chemokines, and a DNA encoding the same. Further, itprovides an antibody, an antisense sequence inhibiting the expression ofthe DNA encoding the above-described protein and a probe consisting of asequence complementary to the above-described DNA. Furthermore, itprovides a method of identifying a substance which inhibits theassociation of the above-described protein with the intracellularC-terminal domain of the receptor by using the protein.

(1) Discovery of Novel Protein

The present inventors have confirmed for the first time the presence ofintracellular signal transduction-regulating polypeptide associatingwith the intracellular C-terminal domain of a receptor CCR2, which isone of G protein-coupled receptors for chemokines, and the DNA encodingthe same, i.e., discovery of a novel intracellular signal transductionmechanism. This protein can be obtained by searching for a moleculespecifically binding to an intracellular domain following the7-transmembrane part of the receptor with the use of the two-hybridmethod, thus obtaining its partial sequence, constructing oligoprimersbased on this sequence, and then cloning the full-length cDNA by usingthe RACE method.

More specifically speaking, a cytoplasmic protein directly associatingwith the intracellular C-terminal domain sequence of the receptor CCR2(the sequence occurring in the 309- to 360-residues) carried by humanmyelomonocytic leukemia can be obtained. Among several clones thusobtained, one contains a cDNA (clone 19 represented by SEQ ID NO:38)encoding a novel polypeptide having a sequence consisting of 156 aminoacids (SEQ ID NO:37). Based on this sequence, oligoprimers areconstructed and a full-length cDNA (clone 1, SEQ ID NO:19) is cloned bythe RACE method. The full-length cDNA (clone 1) thus obtained isamplified by the PCR method and then inserted into a plasmid vectorpcDNA3 (manufactured by Invitorgen) to thereby construct an expressionvector. Next, this expression vector is transferred into HEK293 cells(ATCC/CRL-157) to give a transformant. Expression of FROUNT protein 1(SEQ ID NO:1) is detected by using the Western blotting method.

(2) Structural Characteristics of the Protein

The thus obtained protein having the amino acid sequence represented bySEQ ID NO:1 and associating with the intracellular C-terminal domain ofthe receptor CCR2 (the protein originating in clone 1 which will be alsocalled FROUNT protein 1 hereinafter) has a sequence consisting of 656amino acids and its molecular weight is 79 KDa. Its structuralcharacteristics reside in containing a leucine zipper structure known asplaying an important role in the association between proteins, 4tyrosine-based motives and 4 dileucine motives. FIG. 5(a) shows theKyte-Doolittle hydropathy plot of this protein. When examined byhomology searching, it is clarified that none of known human genes orproteins has a sequence similar to the above-described protein.

(3) Biological Properties

The biological properties of the obtained FROUNT protein 1 confirmed bythe present inventors are as follows.

(1) When observed under a fluoromicroscope, FROUNT protein 1 bindsspecifically to a chemokine receptor CCR2 or CCR5 expressed in HEK cellsin response to stimulation with the chemokine CCL2 and induces theclusterization of the receptor. It is found out that the chemokinereceptors CCR2 and CXCR4 activate chemotactic signal cascades in THP-1cells and human monocytes while FROUNT protein 1 associates with theintracellular C-terminal domain (SEQ ID NO:41) of the receptor CCR2 butnot with that of CXCR4 (FIG. 1). The interaction between FROUNT protein1 and the receptor CCR2 is confirmed by the binding in vitro and acoimmunoprecipitation assay with the use of Myc-tagged and HA-taggedrecombinant FROUNT protein 1 (FIG. 2). Although the above-described12-Pro-C-terminal domain is important in chemotaxis, it is reported thata mutant derived from the sequence by substitution of serine at the317-position and threonine at the 325-position each by alanine (mutantwith 12 residues) is inactive (Document 28). The relationship betweenFROUNT protein 1 and this mutant consisting of the 12 residues isexamined. The association activity of FROUNT protein 1 completelydisappears in the case of removing these 12 residues form the receptorCCR2 but the association activity is conserved in the case of removing ashorter sequence in the C-terminal side of the 12 residue. FROUNTprotein binds to the above-described mutant with the 12 residues. Thesefacts seemingly indicate that FROUNT protein 1 would associate with somepartial sequence in the sequence of the intracellular C-terminal domain(SEQ ID NO:41) of the receptor CCR2 and exerts a function as a regulatorparticipating in the receptor CCR2-dependent chemotaxis.

(2) When transfected cells with lessened expression of FROUNT protein 1therein are established in a cultured cell system with the use of theantisense method, various intracellular signal transduction pathways(cell migration, calcium mobilization, receptor clusterization andreceptor internalization) are lowered. In the case where FROUNT protein1 is coexpressed in the above-described cell system, however, nolowering is observed. In the antisense cell system, cell migrationactivity of CCR5 due to stimulation with a ligand RANTES is lowered.Since these phenomena are not observed in vector control cells or in thecase of stimulating with another chemokine SDF-1, it is confirmed thatFROUNT protein 1 is a molecule which specifically controls CCR2 andCCR5. When the interaction between FROUNT protein 1 and theintracellular C-terminal domain of these receptors is blocked,therefore, responses of the receptor to external stimulation are lost.

(3) Moreover, a mouse with decreased FROUNT protein 1 in bonemarrow-origin leukocytes is established and thioglycolate-inducedmacrophage infiltration ability of this mouse is evaluated. As a result,it is observed that this mouse shows lowered macrophage infiltrationability compared with a control mouse. This fact suggests that thephenomena clarified in the cultured cells reflect these phenomena at theindividual mouse level under physiological conditions. As these resultsshow, it is clarified through the examinations both in vitro and in vivothat the protein obtained by the present invention is a molecule whichplays an important role in the CCR2- and CCR5-mediated monocyte andmacrophage migration.

(4) Other Characteristics

As the results of database (Blast) homology searching, it is revealedthat there are sequences highly homologous with the DNA sequenceencoding FROUNT protein 1 in mouse and Drosophila melanogaster(AAF577B5) as well as in Caenorhabditis elegans (T24318) and yeasthaving no leukocyte (FIG. 5(b)). This fact that the DNA sequenceencoding FROUNT protein 1 is conserved even in primitive organismssuggests that this protein would participate in fundamental lifephenomena in various organisms over a broad range.

(5) Meanings of the Novel Protein

As the detailed acquisition process and biological properties asdescribed above clearly indicate, it is considered that FROUNT protein 1associates with the intracellular C-terminal domains of the chemokine Gprotein-coupled receptors CCR2 and CCR5 and thus participates in themechanism of controlling the G protein switching system. That is to say,it seems that FROUNT protein 1 is a polypeptide which regulates theintracellular signal transduction, acts on the intracellular C-terminaldomain of a receptor together with G protein and thus participates inthe mechanism of controlling the function of the receptor. Since therehas been known neither a chemotactic signal transduction mechanismspecific to an individual chemokine receptor nor a protein capable ofcontrolling the mechanism, it can be concluded that the presence such aprotein per se has been clarified for the first time by the presentinventors.

Based on the fact that the DNA sequence of FROUNT protein 1 is conservedeven in the nematode and yeast having no leukocyte, there is a highpossibility that FROUNT protein 1 and FROUNT-like proteins associatingwith the intracellular C-terminal domains of cell membrane receptorsCCR2 and CCR5 to chemokines would control signal transduction systemsfrom individual receptors not only in other leukocyte chemotacticfactors but also in hormones and cell membrane receptors such asneurotransmitter molecules.

(6) Protein According to the Present Invention and DNA Encoding the Same

Thus, the protein according to the present invention and the DNAencoding the same are primarily an intracellular signaltransduction-regulating polypeptide associating with the intracellularC-terminal domain of a G protein-coupled receptor CCR2 for chemokineCCL2 and a DNA encoding the same, and an intracellular signaltransduction-regulating polypeptide associating with the intracellularC-terminal domain of a G protein-coupled receptor CCR5 for chemokinesCCL's 3, 4 and 5, and a DNA encoding the same. More specificallyspeaking, the protein according to the present invention and the DNAencoding the same are a polypeptide having the amino acid sequencerepresented by SEQ ID NO:1 and a DNA encoding the same. Stillspecifically speaking, the DNA has the sequence represented by SEQ IDNO:20.

(7) Clones Other than Clone 1 and Peptides Corresponding Thereto

In screening a THP-1-origin cDNA library by the plaque hybrid method, aclone (clone 1) of FROUNT protein 1 and another clone 2 (SEQ ID NO:20)are obtained. By examining amino acid sequences deduced based on thebase sequences thereof, it is clarified that human FROUNT involves atleast two molecular types, i.e., a sequence consisting of 656 amino acidresidues (a-type, clone 1) and another one consisting of 630 amino acidresidues (β-type, clone 13) differing from each other exclusively in theC-terminal part. Further, cloning is carried out by the PCR method andthus at least 18 splicing mutants of the α- and β-types represented bySEQ ID NOS:1 to 18 are identified. Each of these clones 1 to 18 has anN-terminus starting with ATG and a C-terminus stopping with atermination codon (TAG, TGA, TAA). It is anticipated that these clonesrespectively have the amino acid sequences represented by SEQ ID NOS:1to 18. Polypeptides corresponding to these clones 2 to 18 will bereferred to as FROUNT proteins 2 to 18 hereinafter. The clones 2 to 18are in common to the clone 1 at least in the N-terminal sequence and,moreover, have common sequences in other parts. Based on these facts, itis considered that the clones 2 to 18 have functions either entirely orpartly common to clone 1. It is also considered that clones 2 to 18 areusable as probes for detecting FROUNT proteins 1 to 18. Furthermore,these polypeptides are usable as antigens for constructing antibodiesand so on.

(8) Gene Structures of FROUNT Proteins 1 to 18

As the results of human genome database (NCBI, Blast) searching, it isidentified that the FROUNT gene clone 1 is encoded as 19 exons in the17th chromosome (in the neighborhood of D17S785 and D17S1352) similar toCCL's 2, 3 and 5. In the FROUNT gene clone 13 (a β-type splicingmutant), the exons 1 to 16 are the same as those in the FROUNT geneclone 1 but the reading frame in the 17th exon is extended backwardcompared with the FROUNT gene clone 1. It is thus confirmed that theFROUNT gene clone 13 is encoded as 17 exons and the FROUNT gene clone 13differs from the FROUNT gene clone 1 exclusively in the C-terminalstructure.

(9) Functions of FROUNT Proteins

As discussed above, it is clearly understood that FROUNT proteins 1 to18 originate in the same genomic DNA (gDNA) and have one of thefollowing functions.

-   -   1. By associating with the intracellular C-terminal domain of        the receptor CCR2 and thus regulating the intracellular signal        transduction, controlling the function of the receptor CCR2 to        thereby promote or inhibit cell migration, calcium mobilization,        receptor clusterization, receptor internalization and so on.    -   2. Providing, as an antigen, an antibody against a FROUNT        protein and being available as a probe for detecting the FROUNT        protein.

That is, FROUNT protein 1 has the above function 1 while FROUNT proteins2 to 18 have at least the above function 2. The term “FROUNT protein” asused herein means a protein having one of the above functions 1 and 2.

(10) Scope of the FROUNT Protein According to the Present Invention

All of the DNA sequence data obtained by sequencing the DNA molecules inthe present invention are obtained by using an automatic DNA sequencer(Model ABI377 manufactured by Applied Biosystems) and the amino acidsequences of the polypeptides according to the present invention arededuced based on the translation of the DNA sequences thus determined.As widely known in this technical field, there is a possibility thateach of the DNA sequences thus determined by using the automatic DNAsequencer has some errors. Therefore, each DNA sequence thus determinedshould be regarded as having typically at least 90% identity, stilltypically at least about 95% to 99.9% identity, to the actual DNAsequence. Accordingly, the present invention involves in its scope a DNAconsisting of a sequence having at least 90% identity to a DNArepresented by any of SEQ ID NOS:19 to 36 and a polypeptide consistingof a sequence having at least 90% identity to a polypeptide representedby any of SEQ ID NOS:1 to 18. The problem concerning errors in DNAsequencing as described above also arises in the sequences 39 and 40(and, moreover, 37 and 38) as will be described hereinafter. In thesecases, therefore, the determined DNA sequences and the amino acidsequences deduced therefrom should be regarded as having typically atleast 90% identity, still typically at least about 95% to 99.9%identity, to the actual DNA sequences too.

(11) Medical Applicability of the Protein According to the PresentInvention

It is reported that CCR2- or CCR5-mediated chemotaxis of monocytes andmacrophages plays important roles in inflammation reactions andparticipates in chronic inflammatory diseases such as arteriosclerosisand autoimmune diseases. Furthermore, it is known that these chemokinereceptor molecules are essentially required in cell entry of humanimmunodeficiency virus (HIV). Thus, it is considered that chemokinereceptor signal controllers, molecules carrying a part of a FROUNTprotein, association-inhibiting molecules or antisenses are usable inpreventing and treating these inflammatory diseases and infections suchas AIDS (acquired immunodeficiency syndrome) induced by HIV and so on.Namely, these molecules are expected as being useful as novel targetsfor establishing therapeutic methods.

FROUNT protein 1 exerts an effect of promoting the CCL2-CCR2 pathway ofmonocytes and macrophages, while its inhibitor, antibody and antisenseexert an inhibitory effect. Therefore, it is expected that FROUNTprotein 1 and its gene are usable in treating diseases caused bylowering in the functions of monophages and macrophages in the CCL2-CCR2pathway, for example, as immunoenhancers, self-defensive reactionpromoters or compositions for treating infectious diseases. On the otherhand, it is expected that the inhibitor, antibody and antisense thereofare usable in treating diseases caused by hyper-reactions in theCCL2-CCR2 pathway, for example, as compositions for treatingatherosclerosis, chronic glomerulonephritis and multiple sclerosis,immunomodulators or antiallergic agents.

Since molecules highly homologous with FROUNT protein are observed evenin the nematode and yeast having no leukocyte, there is a sufficientlyhigh possibility that FROUNT-like proteins would control signaltransduction systems from individual receptors not only in otherleukocyte chemotactic molecules but also in hormones and Gprotein-coupled receptors such as neurotransmitter molecules. Namely,these FROUNT-like proteins are expected as contributing to the provisionof clinical targets over a wide range beyond the field in whichchemokines act. A method of screening a novel agonist and antagonistwith the use of, as an indication, the association of a Gprotein-coupled receptor with a signal-controlling molecule orclusterization of these molecules in response to ligand stimulation ishighly useful.

For example, a compound capable of inhibiting ligand stimulation isscreened by stimulating cells, wherein a FROUNT protein fused with afluorescent protein and receptor(s) CCR2 and/or CCR5 are forciblyexpressed, with combinations of individual ligands with variouscompounds and using clusterization of the FROUNT protein thus induced asan indication. It is found out that this method might be available as anovel system by which a compound controlling signaling of the receptorCCR2 or CCR5 can be easily screened.

FROUNT protein 1 exerts an effect of promoting the CCL2-CCR2 pathway ofmonocytes and macrophages, while its inhibitor, antibody and antisenseexert an inhibitory effect. An association domain peptide has acompetitive effect to FROUNT protein 1 to thereby inhibit its activity.Similar results are obtained by directly transferring the associationdomain peptide into a cell or transferring a gene fragment thereof intoa cell and forcibly expressing therein. Accordingly, use can be made ofFROUNT protein 1, its gene, an inhibitor, an antibody and an antisensethereof, the association domain peptide and an antibody, a gene encodingthe association domain peptide, an antisense thereof, etc. in preventingand treating chronic inflammatory diseases and infectious diseaseswherein the CCL2-CCR2 pathway and the CCL3, 4, 5-CCR5pathways ofmonocytes and macrophages participate.

It is expected that peptides having an association domain sequence,among FROUNT proteins 2 to 18, are expected as having a function similarto FROUNT protein 1. On the other hand, peptides having no associationdomain sequence are usable as probes, antigens for acquiring antibodies,and so on. Such a probe or antibody is useful in detecting andquantifying FROUNT protein 1. Moreover, it is expected that an antibodyand a probe selective to each-clone enable quantitative understanding ofthe balance among FROUNT proteins 1 to 18 potentially expressed incells, thereby contributing to the clarification of the relationship todiseases.

(12) Process of Producing the FROUNT Protein According to the PresentInvention

An expression vector can be obtained by connecting a cloned gene of theFROUNT protein according to the present invention to the downstream of apromoter in a vector appropriate for expression. Examples of the vectorinclude plasmids originating in Escherichia coli (for example, pBR322,pBR325, pUC12 and pUC13), plasmids originating in Bacillus strains (forexample, pUB110, pTP5 and pC194), plasmids originating in yeasts (forexample, pSH19 and pSH15), bacteriophages such as λ phage and animalviruses such as retrovirus and vaccinia virus.

To express the gene, a promoter is further connected to the upstream.The promoter usable therefor may be an arbitrary one so long as it isadequate for the host to be used in the gene expression. In the case ofusing E. coli as the host, for example, use may be made of trp promoter,lac promoter, recA promoter, λPL promoter, lpp promoter and so on. Inthe case of using a host belonging to the genus. Bacillus, use may bemade of SP01 promoter, SP02 promoter, penP promoter and so on. In thecase of using a yeast as the host, use may be made of PH05 promoter, PGKpromoter, GAP promoter, ADH promoter and so on. In the case of using E.coli as the host, it is particularly preferable to use trp promoter orλPL promoter. In the case of using an animal cell as the host, it ispreferable to use an SV40-origin promoter, a retrovirus promoter, etc.and an SV40-origin promoter is still preferable.

Using the vector thus constructed, a transformant is prepared. As thehost, use can be made of, for example, E. coli, a Bacillus strain, ayeast, an animal cell and so on. Examples of E. coli include E. coliK12DH1 (Proc. Natl. Sci. USA 60:160 (1968)), E. coli M103 (Nucleic AcidsResearch 9:309 (1981)), E. coli JA221 (J. Mol. Biol. 120:517 (1978)), E.coli HB101 (J. Mol. Biol. 41:459 (1969)), E. coli C600 (Genetics 39:440(1954)) and so on. Examples of the Bacillus strain include Bacillussubtilis MI114 (Gene 24:255 (1983)), Bacillus subtilis 207-21 (J.Biochem. 95:87 (1984)) and so on. Examples of the yeast includeSaccaromyces Cerevisiae strains AH22R, NA87-11A, DKD-5D and so on.Examples of the animal cell include COS-7, Vero, CHO, mouse L cell,human FL cell and so on.

E. coli is transformed in accordance with, for example, a methoddescribed in Proc. Natl. Acad. Sci. USA 69:2110 (1972) or Gene 17:107(1982). A Bacillus strain is transformed in accordance with, forexample, a method described in Molecular & General Genetics 168:111(1979). A yeast is transformed in accordance with, for example, a methoddescribed in Proc. Natl. Acad. Sci. USA 75:1929 (1978). An animal cellis transformed in accordance with, for example, a method described inVirology 52:456 (1973). It is appropriate to culture the obtainedtransformant in a liquid medium by a method commonly known in the art.

The FROUNT protein can be separated and purified from the culture byharvesting the microbial cells or animal cells from the culture by aknown method, suspending the cells in a buffer solution containing aprotein degenerating agent such as guanidine hydrochloride, disruptingthe cells by ultrasonication, lysozyme-treatment, freezing-thawing, etc.and then collecting the supernatant by centrifugation. Next, the FROUNTprotein is purified and isolated from the supernatant by using, forexample, salting out, precipitation from a solvent, dialysis,ultrafitratin, gel filtration, SDS-polyacrylamide electrophoresis,ion-exchange chromatography, affinity chromatography, reversed-phasehigh performance liquid chromatography, isoelectric focusing or acombination of these procedures.

(13) Method of Administering the Full-Length DNA According to thePresent Invention or a Part Thereof

A required DNA can be transferred into a cell by applying a known methodhaving been already established. Typical examples of such methodsinclude a method which comprises integrating the DNA into anadenovirus-origin vector or a retrovirus-origin vector and administeringas a plasmid and another method of administering as a liposomepreparation.

A liposome is a closed vesicle made of a lipid bilayer membrane andhaving an aqueous layer therein. It is known that this lipid bimolecularmembrane structure is closely similar to a biomembrane. Examples of thephospholipid to be used in producing the liposome preparation accordingto the present invention include phosphatidylcholines such as lecithinand lysolecithin, acidic phospholipids such as phosphatidylserine,phosphatidylglycerol, phosphatidylinositol and phosphatidic acid,phospholipids derived therefrom by substituting acyl group by lauroylgroup, myristoyl group, oleoyl group, etc., shingophospholipids such asphosphatidyl ethanolamine and sphingomyelin and soon. It is alsopossible to add cholesterol, etc. thereto. It is also possible toproduce a liposome from natural materials such as lipids usuallyoccurring in cell membrane by a method commonly known in the art. Aliposome preparation containing the FROUNT protein gene according to thepresent invention can be produced by, for example, suspending a thinphospholipid membrane having been purified in a solution containing theFROUNT protein gene and subjecting to ultrasonication.

The liposome preparation containing the FROUNT protein gene according tothe present invention may be in the form of a membrane-fused liposomeprepared by fusing with an appropriate virus or the like. In this case,it is preferred that the virus has been inactivated by using, forexample, UV rays. As a particularly preferable example of themembrane-fused liposome, a membrane-fused liposome fused with Sendaivirus (hemagglutinating virus of Japan; HVJ) maybe cited. Thismembrane-fused liposome can be produced by a method described in J.Biol. Chem. 266(6), 336-3364 (1991). For example, an HJV-fused liposomepreparation can be prepared by mixing purified HJV having beeninactivated by UV-irradiation with a suspension of liposomes containinga FROUNT protein gene vector, gently stirring the mixture and thenremoving the unbound HJV by the sucrose density gradient centrifugationmethod. Moreover, the gene transfer efficiency into a cell can beelevated by binding a substance having an affinity for the cell (forexample, an antibody, a ligand to a receptor, etc.) to the liposome.

(14) Antisense

An antisense which inhibits the expression of the FROUNT protein in acell includes antisense nucleic acids (RNA or DNA) causing inhibition inthe step of transcription, inhibition in the step of RNA processing,inhibition in the step of membrane permeation of RNA and inhibition inthe step of translation. Furthermore, a DNA producing an antisense RNAin a cell can be used with the purpose of inhibition in the presentinvention. Examples of the antisense nucleic acid molecule according tothe present invention include a nucleic acid molecule complementary tothe sense nucleic acid encoding FROUNT protein, a nucleic acid moleculecomplementary to the regulatory domain of genomic DNA, a nucleic acidmolecule complementary to an mRNA sequence and so on.

As an example of the antisense nucleic acid molecule according to thepresent invention, the full-length of the RNA sequence represented bySEQ ID NO:39 or a part thereof may be cited. The RNA sequencerepresented by SEQ ID NO:39 shows an antisense strand of the siteencoding the C-terminal sequence consisting of 57 amino acid residues inFROUNT protein 13. It is confirmed that an antisense nucleic acidderived therefrom brings about reduction in mRNA in cells not only inFROUNT gene 13 but also in FROUNT gene 1.

It is not always required that the antisense nucleic acid moleculeusable herein is complementary to the entire coding domain. Namely, itmay be an oligonucleotide which is complementary to a part of the codingor non-coding domain of mRNA or a part of a genomic DNA regulatorydomain. The length of the antisense oligonucleotide can be selected fromamong, for example, 5, 10, 15, 20, 30, 40 ad 50 nucleotides.

The antisense nucleic acid according to the present invention can beconstructed by chemical synthesis or enzymatic ligation which has beenknown in the art.

The antisense nucleic acid according to the present invention can beadministered in the form of an RNA molecule. Alternatively, it may beadministered as a DNA molecule capable of expressing the RNA molecule ina cell. For example, the full length of the RNA sequence represented bySEQ ID NO:39 or a part thereof may be administered. It is also possiblethat the full length of the DNA sequence represented by SEQ ID NO:40,which corresponds to the above sequence, or a part thereof isadministered to thereby allow the expression of the corresponding RNA ina cell.

As an example of the administration route of the antisense nucleic acidmolecule according to the present invention, it may be directly injectedinto a tissue site in the form of such a liposome preparation asdescribed above or a plasmid having the antisense nucleic acid moleculeintegrated into a known virus vector. It is also possible that theantisense nucleic acid molecule is modified so as to target a specifictissue and then systemically administered. For example, the antisensenucleic acid molecule can be modified so as to target a selected cell ortissue by binding a peptide or an antibody binding to a cell surfacereceptor or a cell surface antigen. The antisense nucleic acid moleculeaccording to the present invention may be transported into a cell byusing a vector as described above. To achieve a sufficient antisensemolecule concentration in the cell, it is preferable to use a vectorstructure wherein the antisense nucleic acid molecule is under thecontrol of pol II or III promoter.

The present invention further involves a ribozyme for lowering theactivity of the FROUNT protein in a cell. This ribozyme is a catalyticRNA molecule which contains a domain complementary to mRNA and has aribonuclease activity of cleaving the RNA strand. By catalyticallycleaving the FROUNT protein mRNA with the use of the ribozyme, thetranslation of the FROUNT protein can be inhibited. As a typical exampleof the ribozyme usable in the present invention, a hammerhead ribozyme(Haseruhoff and Gerluch, Nature 334:585-591 (1988)) can be cited. Aribozyme specific to a nucleic acid encoding the FROUNT protein can bedesigned based on the DNA sequence represented by SEQ ID NO:19 disclosedin the present description (see, for example, Cech et al., U.S. Pat. No.5,116,742 and U.S. Pat. No. 4,987,071).

The ribozyme can be transported into a cell by directly injection.Alternatively, the transportation can be also made by integrating theribozyme in the form of the corresponding DNA into an inactivatedretrovirus vector, transforming a cell with it and then expressing theribozyme RNA in the cell.

(15) Antibody

The polypeptides represented by SEQ ID NOS:1 to 18, fragments thereof oranalogs thereof are usable as immunogens for producing antibodiesimmunospecific respectively to FROUNT proteins. An antibody against apolypeptide according to the present invention can be obtained byadministering the polypeptide or an epitope-carrying fragment or analogthereof to an animal (preferably a nonhuman animal such as rabbit, goator mouse) in a conventional manner. In preparing the immunogen, use canbe made of an adjuvant or a similar immunostimulant. To produce amonoclonal antibody, it is possible to employ an arbitrary technique bywhich an antibody produced by continuous cell culture can be provided.Examples of such techniques include the hybridoma technique (G. Kohleret al., Nature (1975) 250:495-497), the human B cell hybridoma technique(Kozbor et al., Immunology Today (1983) 4:72), the EBV hybridomatechnique (Cole et al., MONOCLOAN ANTIBODIES AND CANCER THERAPY, p.77-96, Alan R. Liss (1985)) and so on.

The antibody against the FROUNT protein according to the presentinvention can be used in, for example, quantifying or detecting theFROUNT protein. If necessary, the antibody can be labeled with a marker.

(16) Method of Screening Inhibitor

In association with the intracellular localization due tointernalization of receptors CCR2 and/or CCR5 stimulated with a ligand(clusterization), the FROUNT protein according to the present invention,in particular, the FROUNT protein having a function of associating withthe intracellular C-terminal domain of receptor(s) is localized togetherwith the receptor(s) in the cell. By examining the occurrence of theintracellular localization of a marker-labeled FROUNT protein,therefore, the presence or absence of interaction between thereceptor(s) and the ligand can be checked. That is to say, an agonistinhibitor against the receptor(s) CCR2 and/or CCR5 can be identified byforcibly expressing a marker-labeled FROUNT protein in a cell, furtherexpressing the receptor(s) CCR2 and/or CCR5 therein, treating the cellwith an agonist to CCR2 and/or CCR5 and a ample which is a candidate foran inhibitor, observing whether or not the clusterization of thereceptor(s) is induced, and thus judging whether or not the candidatehas an inhibitory effect on the agonist. As the labeling agent, use maybe made of any marker so long as it has no cytotoxicity and does notinhibit the activity of the FROUNT protein. Use can be made of anappropriate substance selected from among various fluorescent proteinssuch as a green fluorescent protein, a red fluorescent protein, a bluefluorescent protein and a yellow fluorescent protein which are availablefrom CLONTECH Laboratories, Inc. (USA). Among all, it seems preferableto employ a red fluorescent protein which is highly distinguishable fromthe background of the cell. Such a fluorescent protein can be easilyexpressed in the state of being fused with the FROUNT protein by aconventional method.

By combining fluorescent proteins with different colors, clusterizationcan be observed depending on color change, which facilitates thedetection. For example, the intracellular localization of FROUNT protein1 fused with a green fluorescent protein and the intracellularlocalization of CCR2 protein fused with a red fluorescent protein areloaded and superposed by using a fluorescent microscope. Thus, thecolocalizatoin of the green fluorescence and the red fluorescence can bevisualized and quantified as yellow fluorescence.

The green fluorescent protein is a protein consisting of 238 amino acidresidues and emitting green light (509 nm) when irradiated with light at350 to 490 nm. It requires neither any other protein, substrate norauxiliary factor for the light emission. Because of being well expressedin various cells as a soluble light-emitting protein, this gene is usedas a reporter gene. By substituting serine at the 65-position of thisprotein by alanine, leucine, cysteine or threonine, its light emissionefficiency can be considerably elevated. These derivatives are alsousable in the present invention.

As the red fluorescent protein, yellow fluorescent protein and bluefluorescent protein which are obtained through mutation of the greenfluorescent protein gene, genes and proteins are marketed from, forexample, CLONTECH Laboratories, Inc. (USA).

Known methods for detecting the occurrence of stimulation with a ligandinclude a method of detecting cell chemotaxis, a method of detectingCa⁺⁺ produced in a cell, and so on. In these methods, however, a largenumber of cells are needed or the detection procedures are troublesome.According to the method of the present invention, in contrast thereto,judgment can be made merely by using several cells and microscopicallyobserving the cells after the stimulation. Namely, in the case where themarker is localized in the cells, it is judged that the ligand has notbeen inhibited. In the case where the marker is scattered in the cells,it is judged that the ligand has been inhibited. Since screening can becarried out with the use of a small number of cells, it can be concludedthat this method is highly advantageous in treating a large number ofcells.

The new screening method with the use of a FROUNT protein thusestablished has been further improved to thereby provide a novelscreening method which has never been known so far. That is, a method ofscreening an inhibitor by taking advantage of the phenomenon thatclusterization of a receptor and a signal transducing molecule on cellsurface is induced in response to stimulation with a ligand. There aremolecules other than FROUNT proteins which couple or associate with theintracellular terminus of receptors. Examples of such molecules includeG proteins, etc. It is also known that not only chemokines but alsohormones act on receptors to cause clusterization. Thus, it is intendedto propose a method of screening an antagonist which comprises preparinga cell having a receptor undergoing clusterization in response tostimulation with an agonist and a marker-labeled molecule coupling orassociating with the intracellular C-terminal domain of the receptorhaving been forcibly expressed therein, treating the cell with anagonist and a specimen containing a candidate for an antagonist, thenobserving whether or not the clusterization of the marker is induced inthe cell, and a cell therefor. Typical examples of the receptor includetransmembrane receptors interacting with chemokines or cytokines, inparticular, 7-transmembrane receptors, I type cytokine receptors,tyrosine kinase receptors, serine/threonine receptors and so on. In the7-transmembrane receptors, examples of molecules coupling or associatingwith the intracellular C-terminal domain of the receptors include FROUNTproteins as well as G proteins, GRKs, Arrestins and so on. In such acase, the above-described colocalization can be utilized by labelingboth of the receptor and the molecule coupling or associating with theintracellular C-terminal domain of the receptor with proteins emittinglights in different colors.

As an application example of the method of screening an inhibitor withthe use of the phenomenon that clusterization of a receptor on cellsurface is induced in response to stimulation with a ligand, a methodwith the use of a chimeric receptor may be cited. Namely, a method ofscreening an agonist inhibitor with the use of a chimeric receptor cellcomprising: forcibly expressing a chimeric receptor, which is obtainedby integrating a DNA sequence encoding the full length or a part of aFROUNT protein-association sequence in the intracellular C-terminaldomain of the receptor CCR2 (more specifically speaking, the amino acidsequence represented by SEQ ID NO:41) into the intracellular C-terminaldomain of the DNA sequence of a desired receptor, in a cell appropriatefor the desired receptor; constructing a cell having a labeled FROUNTprotein and the chimeric receptor by forcibly expressing amarker-labeled FROUNT protein (for example, a FROUNT protein fused witha green fluorescent protein) in the above-described cell; treating thechimeric receptor cell with an agonist to the receptor and a candidatesubstance (a specimen) for an agonist inhibitor; and then observingwhether or not the localization of the marker is induced in the chimericreceptor cell. In this method, the chimeric receptor and themarker-labeled FROUNT protein can be easily expressed in the cell byusing plasmids obtained by integrating DNA sequences respectivelyencoding the same into known expression vectors. In this case, theabove-described colocalization can be utilized by labeling both of thechimeric receptor and the FROUNT protein with proteins emitting lightsin different colors. The present invention further involves in its scopesuch plasmids and cells transformed by these plasmids.

Furthermore, the present invention involves in its scope a method ofidentifying an intracellular signal transduction pathway inhibitordepending on the binding of a FROUNT protein to a receptor with the useof the characteristics of the FROUNT protein which comprises using thebinding activity of the FROUNT protein to the receptor as an indicationand screening a substance inhibiting the binding activity.

According to the screening methods of the present invention as describedabove, an inhibitor can be accurately screened by using an extremelysmall number of cells for a desired combination of a receptor with anagonist.

By combining the phenomenon of the association of a FROUNT protein withthe C-terminal domain of a receptor with a labeling agent as in thepresent invention, clusterization and colocalization can be visualized.Namely, biological phenomena in cells can be more easily grasped,observed and detected directly with eye compared with the existingmethods. Moreover, it is expected that various cells transformed by theabove procedure are widely applicable and usable in detecting cytotoxicsubstances, detecting environmental pollutants, examining cytotoxicityof drugs and so on. It is also possible to judge whether or not aspecimen contains a cytotoxic substance by treating a cell having beentransformed by the procedure according to the present invention with thespecimen, then treating it with an agonist to the receptor carried bythe cell and observing whether or not clusterization or colocalizationis induced. Use of the cells according to the present invention makes itpossible to carry out various detections at a high accuracy with the useof an extremely small number of cells.

(17) Diagnosis and Examination

It is considered that the absence, abnormal amount or abnormality in thesequence of a protein or nucleic acid associating with the intracellularC-terminal domain of a receptor would relate to abnormality inintracellular signal transduction and, therefore, likely affect theextent of efficacy or side effects of drugs. Accordingly, it is expectedthat the examination on the presence or absence of the above-describedabnormalities provide important clues in diagnosing diseases. That is tosay, detection of these factors provides novel means of understandingdisease conditions which has never been available so far. For example, aFROUNT protein can be quantified by using an antibody, while a mutationin a sequence can be examined by applying known procedures, e.g.,determining the sequence of a DNA or RNA fragment collected from aspecimen and comparing it with a normal sequence, or by screening a DNAsequence contained in a specimen with the use of a probe having asequence complementary with DNA sequence encoding a normal FROUNTprotein and examining whether or not complete hybridization arises.

Now, the present invention will be described in greater detail byreferring to the following EXAMPLES, though it should be understood thatthe invention is not restricted thereto.

EXAMPLE 1 Isolation of Clone 19

(1) Preparation of THP1-Origin cDNA Library Fusing with TranscriptionalActivation Domain of Yeast Gal4

RNA was extracted form THP-1 cells (ATCC:TIB-202) by using the guanidineisothiocyanate method (Chirgwim et al., Biochemistry 18, 5294 1978).From this RNA, poly(A)RNA was purified by oligo dT cellulose columnchromatography (Aviv & Leder, Proc. Natl. Acad. Sci. USA 69:1408(1972)). By using the thus obtained poly (A) RNA as a template, aTHP1-origin cDNA library fusing with the transcriptional activationdomain of yeast Gal4 was prepared by using a pACT2 vector (CLONTECHLaboratories, Inc.,) in accordance with the method of Okayama and Berg(Okayama & Berg, Mol. Cell. Biol. 2:161 (1982); ibid., 3:280 (1983)).Then this cDNA library was transferred into E. coli DH10B and plasmidDNA was extracted by the alkali method (Birnboim, H. C. & Doly, J.,Nucleic Acids Res. 1:1513 (1979)). Thus, a cDNA library consisting ofabout 2×10⁵ clones was prepared with the use of E. coli DH10B as thehost.

(2) Construction of Vector Expressing Fused Protein Composed ofDNA-Binding Domain of Yeast GAL4 and Intracellular C-Terminal Sequence(The Residues 309 to 360) of Human CCR2

A bait fragment containing the amino acid residues 309 to 360 in theintracellular C-terminal side of human CCR2b was amplified by apolymerase chain reaction (PCR) method by pCMGS-CCR2b in accordance withJ. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.(1989) with the use of a forward primer 5′GCGAATTCGAGAAGTTCAGAAGGTAT3′(SEQ ID NO:42) and a reverse primer 5′GCGGATCCTTATAAACCAGCCGAGAC(SEQ IDNO:43). The thus amplified fragment was treated with restriction enzymes(EcoRI and BmaHI) and then orientationally cloned into pACT2 (a yeastGal4 DNA binding domain cloning vector containing TRYP gene forselection in a yeast lacking tryptophan biosynthesis (CLONTECHLaboratories, Inc.)). The base sequence of the bait fragment of thisplasmid DNA was amplified by the PCR method with the use of a primerspecific to the adjacent sequence of the vector in accordance with thedideoxynucleotide chain termination method (J. Messing et al., NucleicAcids Res. 9:309, (1981)). Then the base sequence was determined byusing an automatic DNA sequencer (Model ABI377 manufactured by AppliedBiosystems).

(3) Transfer of the Above Vector into Yeast Cells and Isolation of YeastCells Showing Interaction

The THP1-origin cDNA library and bait vector as described above weretransformed into a yeast strain Y190 in accordance with the lithiumacetate/polyethylene glycol transformation protocol (see, Ito et al., J.Bacteriolo. 153:163-168 (1983)). On a synthetic complete (SC) mediumlacking tryptophan, leucine and histidine (SC Trp Leu His) andcontaining 10 mM of 3-aminotriazole (Sigma Chemical Co.), a vectortransformant encoding a library protein interacting with the C-terminalsequence of human CCR2 was selected. Next, the interaction betweenproteins was quantified by using β-galatosidase activity as anindication by the colony lift β-galactosidase filter assay (Breeden andNasmyth, Cold Spring Harbor Quant. Biol. 50:643-650 (1985)) and thusyeast cells showing the interaction occurring therein were isolated.

(4) Isolation of Vector from Positive Yeast Cells, Determination of BaseSequence and Confirmation of Interaction Specificity

Yeast cells positive to protein interaction, which contained a mixtureof a binding domain plasmid with an activation domain plasmid, werere-cultured as an isolated matter in each well of a 96-well microtiterplate. Then, about 10 μl of each isolated matter was dissolved and aninsert in the pACT2 plasmid was amplified by PCR with the use of primersspecific to the adjacent sequences of individual vectors. The basesequence of the insert was isolated and determined by theabove-described method. Thus, the sequence (SEQ ID NO:38) of a gene(clone 19) encoding 156 amino acids was identified and compared withpublicly known sequences by using a “BLAST” program available in publicmediated by National Center for Biotechnology Information (NCBI). Thus,it was confirmed that this clone was a novel gene the function of whichhad never been analyzed. The gene (clone 19) encoding 156 amino acidswas named FROUNT gene and the isolated plasmid clone was namedpACT2-FROUNT. To confirm the binding specificity of the protein (FROUNTprotein) encoded by the FROUNT gene (clone 19), the following two testscommonly employed in the art were first carried out.

In the first test, Y190 cells expressing individual plasmids containingDNA sequences encoding FROUNT protein and receptors CCR5 and CCR2 andsequences encoding FROUNT protein:p53 (CLONTECH Laboratories, Inc.,),FROUNT protein:CXCR4, FROUNT protein:CCR5 and FROUNT protein:CCR2 wereprepared in the same manner. When the proliferating ability andβ-galactosidase activity of these yeast cells were tested, nocombination other than FROUNT protein:CCR2 and FROUNT protein:CCR5showed any proliferating ability or β-galactosidase activity. Thus, itwas confirmed that FROUNT protein is not a “self-activating” protein(i.e., requiring the interaction with a second protein domain to form afunctional activated complex) and that FROUNT protein has bindingability specific to the receptors CCR2 and CCR5 (FIG. 1). In FIG. 1which shows the results of a yeast two-hybrid assay, GAL4 BD and GAL4 ADrespectively stand for a transcriptional factor binding domain-fusedprotein expression vector and an activation domain-fused proteinexpression vector; BD or AD vector stands for a control (empty) vector;FROUNT, p53, SV40 T-antigen, CCR5, CXCR4 and CCR2 stand for transferredgenes; each numerical value in parentheses indicates the number ofinserted amino acid residues; -LDH stands for proliferating ability inthe deficient medium; β-Gal activity stands for β-galactosidaseactivity; and + and − stand respectively for the presence and absence ofthe activity.

In the second test, coding sequences of pAS2-1-CCR2b and pAS2-1-p53 wereamplified by PCR using a T7 promoter, a 5′ primer encoding myc epitope:5′AAAATTGTAATACGACTCACTATAGGGCGAGCCGCCACCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGGTATCGCCGGTATTG 3′ (SEQ IDNO:44) and a 3′ primer originating in p AS2-1: 5′CAGCTATGACCATGATTACGC3′(SEQ ID NO:45) to express an epitope-tagged protein. Similarly,pACT2-FROUNT was amplified by PCR using a T7 promoter, a 5′ primerencoding HA epitope: 5′AAAATTGTAATACGACTCACTATAGGGCGAGCCGCCACCATGTACCCATACGACGTTCCAGATTACGC3′ (SEQ ID NO:46) and a 3′ primer originating in pACT2: 3′ACTTGCGGGGTTTTTCAGTATCTACGAT5′ (SEQ ID NO:47). Then,isotope-labeled recombinant proteins were produced in accordance withuser's manual of MATCHMAKER Co-IP Kit (CLONTECH Laboratories, Inc.). Bythe coimmunoprecipitation method using these recombinant proteins, thespecific binding ability between CCR2 and FROUNT was confirmed again(FIG. 2). In FIG. 2 which shows the results of SDS-PAGE analysis incoimmunoprecipitation, myc-CCR2, p53 and HA-FNT stand forisotope-labeled myc or HA-fused recombinant protein; I.P. stands for anantibody employed in the coimmunoprecipitation of each sample; HA mAband Myc mAb stand for antibodies specific for Ha and Myc respectively;HA-peptide and Myc-peptide stand for antigen peptides; and + and −respectively stand for the presence and absence for each sample. Namely,coprecipitation occurred by using the HA- or Myc-specific antibody inthe coexistence of myc-CCR2 and HA-FNT, the coprecipitation disappearedby adding an antigen peptide and no coprecipitation occurred in the caseof myc-p53, thereby indicating specific binding ability.

EXAMPLE 2 Isolation of Full-Length cDNA (Clone 1)

THP-1-origin cDNA prepared by using the same method as in thepreparation of the plasmid cDNA library in EXAMPLE 1 was inserted into aλ phage vector λZIP (GIBCO BRL) to give a phage cDNA library using E.coli DH10B as the host. The phage cDNA library using E. coli DH10B asthe host was sowed on 10 soft agar plates at a density of about 1×10⁵clones per plate. After transferring on a nitrocellulose filter (HATFfilter, MILLIPORE), the library was dissolved in a 0.5 N NaOH solutionand the phage DNA thus exposed and denatured was dried and immobilizedon the filter (Maniatis et al., Molecular Cloning, Cold Spring HarborLaboratory, p. 320 (1982)). On the other hand, the above-describedFROUNT gene fragment (clone 19) was ³²P-labeled by the Nick translationmethod (Maniatis et al., ibid., p. 109) and employed as a probe. Thelabeled probe and the filter having the DNA thus immobilized thereonwere subjected to association in 5× SSPE (0.9 M NaCl 50 mM sodiumphosphate buffer solution (pH 7.4), 5 mM EDTA) containing the labeledprobe, 50% formamide, 5× Denhardt's, 0.1% SDS, 100 μg/ml denaturedsalmon sperm DNA solution (10 ml) at 42° C. for 16 hours. After thecompletion of the reaction, the filter was washed in 2×SSC (1×SSC=0.15 MNaCl, 0.015 M sodium citrate) 0.1% SDS solution at room temperaturetwice each for 30 minutes and then in 1×SSC, 0.1% SDS solution at 68° C.twice each for 30 minutes. After drying the thus washed filter, it wassubjected to radioautogram and a clone reacting with the probe wassearched for.

From the clone thus obtained, phage DNA was extracted by the method ofDavis et al. (Dvais et al., Advanced Bacterial Genetics, Gold SpringHarbor Laboratory (1980)) and the cDNA base sequence of the clonereacting with the probe was determined. Further, a cDNA library forracing was prepared from THP-1 cells in accordance with the protocol of5′ race PCR marathon system (TOYOBO). Using this library, the5′-terminal base sequence of the obtained gene was clarified. Usingoligonucleotides chemically synthesized based on the 5′-terminal and3′-terminal base sequences, human FROUNT gene full-length cDNA wascloned by the PCR method with the use of the THP-1 cell-origin cDNAlibrary as a template. Then the full base sequence was determined byusing an ABI377 sequencer (clone 1).

In accordance with Kozak's anticipation (Kozak, M., Cell 44:283-292(1986)), a potential initiation codon was found out and it was followedby a complete open reading frame encoding a 656 amino acid protein (SEQID NO:1/FROUNT protein 1) having a calculated molecular weight of 79kDa. As the results of a search on Gene Bank, it was found out thatFROUNT protein 1 encoded by the clone 1 gene is a novel one. Throughdatabase motif searching, it was found out that this protein carries aleucine zipper structure, 4 leucine motives and 4 dileucine motivesknown as playing important role in protein-protein binding (FIG. 4 andFIG. 5(c)). From the Kyte-Doolittle hydropathy plot, it was estimatedthat this FROUNT protein is an intracellular protein (FIG. 5(a)). As theresults of a search on human genome database (NCBI, BLAST), it wasidentified that FROUNT genome is encoded as 26 exons in the 17thchromosome (in the neighborhood of D17S785 and D17S1352) similar toCCL's 2, 3 and 5. In homology searching, no known gene or protein havinganalogous sequence was detected in humans.

FIG. 4 shows the amino acid sequence of human FROUNT protein 1 (hFROUNT)wherein the framed part corresponds to FROUNT conserve domain (FCD), theunderlined part corresponds to the part having been isolated by theyeast two-hybrid assay as described above (SEQ ID NO:38), and 4 starsrespectively indicate 4 leucine residues conserved in the leucine zipperdomain.

FIG. 5 shows the results of the Kyte-Doolittle hydropathy plot of humanFROUNT protein 1 wherein the horizontal axis refers to the number ofamino acid residues while the longitudinal axis refers to thehydrophobicity of each amino acid residue.

EXAMPLE 3 Identification of Binding Domain

To search for a site in the human CCR2 C-terminal sequence (the residues309 to 360) essentially required in the binding to FROUNT protein 1,various expression vectors having mutations in the C-terminal sequence(CR2-1 to CR2-7 in FIG. 3) were constructed as in pAS2-CCR2 and thebinding ability to FROUNT protein was examined by using the yeasttwo-hybrid method as described above. As a result, mutants lacking theresidues 329 to 360 in the C-terminus of CCR2 (i.e., CR2-1 to CR2-3 inFIG. 3) sustained the binding ability, while a mutant lacking theresidues 317 to 360 in the C-terminus of CCR2 (i.e., CR2-7 in FIG. 3)showed no ability to binding to FROUNT protein. These results suggestthat, in the intracellular C-terminal domain (the residues 309 to 360)of CCR2, all of the 12 residues (317 to 328) or a part thereof might bea site essentially required in the binding to FROUNT protein 1. It isalso suggested that this site (the residues 317 to 328) essentiallyrequired in the binding agrees with the 12 residues (12 C-terminus;SVFFRKHITKRF/SEQ ID NO:41) in CCR2 having been reported as essentiallynecessary in the activation of the leukocyte chemotactic signal mediatedby the CCL2-CCR2 pathway. Moreover, it is known that the activation ofthe leukocyte chemotactic signal mediated by the CCL2-CCR2 pathway isnever affected by the substitution of serine of the residue 317 orthreonine of the residue 325 in the 12 residues (317 to 328) of CCR2essentially required in the binding by alanine. Thus, the bindingability was also examined in mutants wherein serine of the residue 317or threonine of the residue 325 was substituted by alanine. As a result,it was observed that these CCR2 mutants (CR2-4 to CR2-6 in FIG. 3)sustained the binding ability too.

Based on these results, it has been clarified that the CCR2 siteessentially required in the binding to FROUNT protein 1 completelyagrees with the CCR2 site essentially required in the activation of theleukocyte chemotactic signal mediated by the CCL2-CCR2 pathway. Theseresults provide sufficient evidence for considering that the binding ofFROUNT proteinl to CCR2 affects the activation of the leukocytechemotactic signal mediated by the CCL2-CCR2 pathway. Since the mutantat the residues 309 to 328 alone (CR2-3 in FIG. 3) in the CCR2C-terminal sequence (the residues 309 to 360) sustained the ability tobind to FROUNT protein, this site (the residues 309 to 328) wasidentified as the binding domain of CCR2 to FROUNT protein. FIG. 3 showsCCR2 mutants (CR2-1 to CR2-7) and the results of the yeast two-hybridassay using them as in FIG. 1.

EXAMPLE 4 Comparison of FROUNT Protein Among Species

A cDNA library originating in mouse bone marrow cells was prepared bythe above-described method. Then mouse FROUNT gene was isolated as inthe human FROUNT protein 1 gene (clone 1/SEQ ID NO:19). By using asoftware for sequence comparison (Network Protein Sequence, CLUSTALW),it was clarified that the amino acid sequence deduced from the basesequence of the mouse FROUNT gene was highly homologous with humanFROUNT protein 1 (FIG. 5(b)). As the results of searching for proteinshaving high homology with human FROUNT protein 1 in species other thanhumans and mouse with the use of publicly known database (NCBI, BLAST),FROUNT proteins of Drosophila melanogaster and Caenorhabditis eleganswere identified. These proteins are all in the almost same size, showhigh homology over the entire domains and sustain sequences havingextremely high homology at almost the center of the protein (theresidues 397 to 441 of human FROUNT protein 1). This site was namedFROUNT conserve domain (FCD) (FIG. 5(c)). These facts indicate thathuman FROUNT protein is highly conserved in different species too.

FIG. 5(b) shows comparison of FROUNT protein among species and aschematic model of the domain structure of human FROUNT protein 1. Inthis figure, homology to human FROUNT protein 1 at the amino acid levelin FCD, the upstream domain of FCD or the downstream domain of FCD isgiven in (%). In FIG. 5(c), hFROUNT is a schematic model of the domainstructure of human FROUNT protein 1; 4 Y's represent tyrosine motives; 4L's represent dileucine motives; FCD represents the FROUNT conserveddomain; the underlined part in the CCR2 binding site corresponds to thepart isolated by the yeast two-hybrid assay as described above; 4 starsrepresent 4 leucine residues conserved in the leucine zipper domain; andFNT-D Ab and FNT-A Ab represent each the site employed as an antigen inconstructing a specific polyclonal antibody.

EXAMPLE 5 Identification of FROUNT Protein 1 Gene Splicing Mutant

Using the plaque hybrid method, amino acid sequences deduced from thebase sequences of other clones isolated from the phage library wereexamined. As a result, it was clarified that human FROUNT proteinseemingly involves at least 2 molecular types, i.e., α-type having 656amino acid residues and β type having 630 amino acid residues differingfrom each other exclusively in the C-terminal part. Since FROUNT R-typeDNA was encoded in the neighborhood of the C-terminus of the FROUNTprotein genome gene, it was confirmed that they are splicing mutants. Byusing a TOPO TA cloning system (Invitrogen) with the use ofoligonucleotides which were chemically synthesized based on the 5′- and3′-terminal base sequences of the FROUNT α- and β-types, furthermore,the base sequences of a plural number of clones were determined. As aresult, it was confirmed that insertion or deletion mutants occurred inboth of the FROUNT α- and β-types. Thus, it was clarified that there areat least 18 types of splicing mutants of FROUNT protein 1 in total inthe FROUNT α- and β-types (SEQ ID NOS:1 to 18, SEQ ID NOS:19 to 36).

Human FROUNT clone 2 (α-deletion type (FIG. 8)) is a FROUNT mutanthaving 611 amino acids derived from human FROUNT clone 1 (α-type, 656amino acids in the full-length (FIG. 7)) by deletion of the bases 662 to792 (135 bp). Human FROUNT clones 3 to 12 also belong to the α-deletiontype.

Human FROUNT clone 14 (β-deletion type (FIG. 10)) is a FROUNT mutanthaving 611 amino acids derived from human FROUNT clone 13 (β-type, 630amino acids in the full-length (FIG. 9)) by deletion of the bases 470 to661 (192 bp). Human FROUNT clones 15 and 16 also belong to theβ-deletion type.

Human FROUNT clone 17 (β-insertion type (FIG. 11)) is a FROUNT mutanthaving 518 amino acids derived from human FROUNT clone 13 (β-type, 630amino acids in the full-length (FIG. 9)) by insertion of 90 bp betweenthe bases 1581 and 1582 followed by frame shifting. Human FROUNT clone18 also belongs to the β-insertion type.

In each figure, an underlined part corresponds to the amino acidsequence identical with human FORUNT 1, while **** represents a deletionor insertion site.

EXAMPLE 6 Detection of FROUNT Protein and Distribution Thereof in CellUsing Green Fluorescent Protein-Fused FROUNT Protein 1

(1) Construction of Plasmid Vector for Expressing Green FluorescentProtein-Fused FROUNT in Animal Cell

Human FROUNT protein 1 gene (clone 1) was amplified by the PCR methodwith the use of a 5′-primer: 5′CCCGCTCGAGCTATGTATTTTGACTGGGGTC3′ (SEQ IDNO:48) and a 3′-primer: 5′GCGA ATTCTCAGGAACCTTCCAGTGAGC3′ (SEQ ID NO:49)and then treated with restriction enzymes (XhoI and EcoRI). Afterinserting into the XhoI, EcoRI site of pEGFP Cl (CLONTECH Laboratories,Inc.), the base sequence of the insert was determined by using an ABI377Sequencer. Thus, a plasmid vector PEGFP-FORUNT for expression in animalcells was constructed. This plasmid vector was transformed into E. coliDH5α in accordance with a method reported in Proc. Natl. Acad. Sci. USA69:2110 (1972) and a plasmid DNA for transforming animal cells wasobtained in accordance with the protocol of Max Prep (Qeagen).

(2) Expression of Green Fluorescent Protein-Fused FROUNT in Animal Cell

Human HEK293 cells (ATCC:CRL-157) were grown by monolayer culture in aDMEM medium containing 5% of fetal bovine serum (Falcon size: 100 mm, 5plastic dishes). After replacing the medium with a fresh one of the sametype, the cells were further cultured in a G418-containing medium afterthe transformation. 4 hours after the replacement, calcium phosphate gelcontaining 30 μg/dish of the plasmid pEGFP-C1 (control) or pEGFP-FROUNTDNA was prepared and added to the cells in accordance with a publiclyknown method (Graham et al., Virology 52:456 (1973)) to give pEGFP-C1transformed cells or pEGFP-FROUNT transformed cells respectively. 4hours thereafter, the above pEGFP-C1 transformed cells or pEGFP-FROUNTtransformed cells were treated with glycerol and further cultured in amedium containing 5% of fetal bovine serum. 24 and 48 hours after thetransformation, the cells were stripped off from the dish by usingtrypsin and an EDTA solution. Then the expression of the greenfluorescent protein (GFP) in the pEGFP-C1 and PEGFP-FROUNT transformedcells was confirmed with the use of a fluorocytometer EPICS ELITE ESP(Beckman Coulter) or EPICS XL/XL-MCL system 2 (Beckman Coulter).Further, the localization of the FROUNT protein in the cells wasobserved by a publicly known method with the use of a confocalfluoromicroscope. As a result, it was confirmed that the control eGFPwas observed all over the cells including nucleus while the GFP-fusedFROUNT protein was localized in the cytoplasm other than nucleus (FIG.6(a)).

In order to clarify the interaction between the green fluorescentprotein-fused FROUNT protein 1 and CCR2 in animal cells, a plasmidvector for expressing human CCR2 protein in animal cells was constructedby the same procedure and then transferred into the pEGFP-C1 andpEGFP-FROUNT transformed cells as described above. Then theintracellular localization of the green fluorescent protein was observedbefore and after stimulating with a CCR2 antibody or CCL2 which is aligand to CCR2. In the case of the CCL2 stimulation, immunostaining wasperformed by using a red fluorescent-labeled human CCR2-specificantibody (R&D System Inc.) so as to simultaneously visualize theintracellular localization of the green fluorescent protein-fused FROUNTprotein 1 and the CCR2 protein under a fluorescent microscope. By thissimultaneous visualization of 2 proteins with the use of green and redfluorescent proteins, the binding and neighboring of proteins in theanimal cells could be visualized as yellow fluorescence, i.e.,overlapping of green and red fluorescences (colocalization). As aresult, little colocalization of the green fluorescent protein-fusedFROUNT protein 1 and CCR2 was observed before the CCL2 stimulation but,after the stimulation with the CCR2 antibody or CCL2, colocalization ofthe green fluorescent protein-fused FROUNT protein 1 was found out inassociation with the accumulation and localization of CCR2 (FIG. 6(b)).FIG. 6 presents fluorescent microphotographs of cells having controleGFP (upper) and fluorescent protein-fused FROUNT (lower) transferredtherein. FIGS. 6(a) and 6(b) respectively show micrographs before andafter treating with the CCR2 antibody. The Merge image shows thesimultaneous visualization of green fluorescence and CCR2 (redfluorescence). Similar results were observed in the case of the CCL2stimulation (data not shown).

No such colocalization was observed in the case of stimulatiing withanother chemokine SDF-1 or stimulating cells expressing the control eGFPalone with CCL2. These facts suggest that FROUNT protein 1 would undergoaccumulation and clusterization specifically to CCR2 in association withthe binding of CCL2 to CCR2. When PEGFP-FROUNT transformed HEK293 cellsexpressing CCR5 in the same manner were stimulated with RANTES which isa CCR5 ligand, colocalization of FROUNT protein and CCR5 was similarlyobserved. In the fluorescent microscopic observation, a fluorescentmicroscope system FV300+IX70 (OLYMPUS) and a cooled CCD camera SenSys(Photometrics)+AX80 (OLYMPUS) were used, while Photosphop (Adope) wasemployed in image processing.

EXAMPLE 7 Preparation of FROUNT Protein 1-Specific Polyclonal Antibodyand Detection of Endogenous FROUNT Protein 1

The site common to FROUNT proteins of the α- and β-types in human FROUNTprotein 1 gene (clone 1) was amplified by the PCR method with the use ofa 5′-primer: 5′CGGGATCCGCCATGTATTTTGACTGGGGTC3′ (SEQ ID NO:50) and a3′-primer: 5′GCGAATTCTCATGACAAAATGGAGACCTGGCTGC3′ (SEQ ID NO:51),treated with restriction enzymes (EcoRI and BmaHI) and thenorientationally cloned into the EcoRI, BamHI site of pGEX4T3 (AmershamPharmacia). Then the base sequence of the insert was determined by usingan ABI377 sequencer. Thus, a plasmid vector pGX-FROUNT for expressingglutathione S transferase (GST)-fused protein was constructed. Thisplasmid vector was transformed into E. coli BL21 (DE3) in accordancewith a method described in Proc. Natl. Acad. Sci. USA 69:2110 (972))followed by expression in accordance with a publicly known protocolrecommended by Amersham Pharmacia. Then the GST-fused protein waspurified by glutathione sepharose affinity chromatography. By SDS PAGEand Coumassie staining, it was judged that the accuracy and purity ofthis GST-fused FROUNT both exceeded 95%.

This GST-fused FROUNT protein 1 (100 μg) was mixed with an adjuvant andemployed in subcutaneously immunizing a New Zealand white rabbit in apublicly known time course. After confirming an increase in the titer, aFROUNT protein 1-specific polyclonal antibody was purified from theserum of the animal. The titer of the FROUNT protein 1-specificpolyclonal antibody was confirmed by calibrating the ability to bind tothe GST-fused FROUNT protein 1 with the use of the Western blottingmethod. Endogenous FROUNT protein 1 was detected by a publicly knownimmunostaining method with the use of the above-described specificpolyclonal antibody. It was thus confirmed that the GST-fused FROUNTprotein 1 showed localization similar to the green fluorescentprotein-fused FROUNT protein 1. These results clearly indicate thatFROUNT protein 1 is accumulated specifically in a chemokine receptor andclusterized with the activation of the CCL2-CCR2 pathway or the CCL3, 4or 5-CCR5 pathway.

EXAMPLE 8 Analysis of the Function of FROUNT Protein Using Cell HavingAntisense Transferred Thereinto

(1) Preparation of Antisense Expression Vector and Establishment of CellLine

Partial sequences of a plural number of FROUNT protein genes wereamplified by the PCR method. Then each fragment was inserted in thereverse direction into a retrovirus vector pEGFPMY (Onai, N. et al.,Blood., 96239-247 2074 (2000)) and the base sequence was determined byusing an ABI377 sequencer (FIG. 12). FIG. 12 is a schematic model of theretrovirus vector pEGFPMY wherein Cont stands for a control (emptyvector); hFNT stands for an expression vector of human FROUNT protein 1;AS-22 stands for an antisense expression vector of human FROUNT protein;DN-hFNT stands for a partly deficient FROUNT protein expression vector;LTR stands for a long terminal repeat; gag stands for a structuralprotein; eGFP stands for a green fluorescent protein; and IRES standsfor a ribosomal entry site.

These vectors were transferred into Phoenix cells (Dr. Garry P. Nolan)by the above-described transformation method. After 2 days, cellsupernatant containing the thus produced recombinant retroviruses wascollected. Then HEK293 and THP-1 cells were infected with theserecombinant retroviruses and the infected cells alone were separated andpurified to a purity of 98% or higher by using a cell sorter systemEPICS ELITE ESP (Beckman Coulter) with the expression of eGFP geneencoded by the retroviruses as an indication. From the recombinantretrovirus-infected cells thus purified, RNA was collected by a publiclyknown method. Using ABI7700, FROUNT mRNA was compared and quantified. Asa result, a cell line wherein the expression of about 90% of FROUNT mRNAwas inhibited could be established in association with the transfer ofan antisense expression vector (AS-22), which was obtained by amplifyinga fragment by the PCR method with the use of a 5′-primer:5′GCGGATCCTCAAATCAAGCAGTGTTTGTC3′ (SEQ ID NO:52) and a 3′-primer:5′CGGGATCCGCCATGCTTTTGGAACAGAAACAGGTG3′ (SEQ ID NO:53), treating withBamH1 and inserting into pMY, into HEK293 cells by the above-describedmethod (FIG. 13). In quantifying mRNA, use was made of a quantitativeTR-PCR system ABI PRISM7700 system (PE Applied Biosystems) and a Taq ManProbe: 5′CCTCGGTCTTTCTGGATGACTCTGCT3′ (SEQ ID NO:54), a 5′-primer:5′CAGCCATGATGCTCAGTGA3′ (SEQ ID NO:55) and a 3′-primer:5′TGGTCTCTATGTCATCATCCTG3′ (SEQ ID NO:56) were synthesized and employed.

Moreover, a remarkable lowering was observed in the detection ofintracellular FROUNT protein level by the Western blotting method withthe use of a FROUNT protein-specific antibody and the immunostainingmethod (FIGS. 13 and 14). FIG. 13 shows the results of thequantification of human FROUNT mRNA (upper a) and FROUNT protein (lowerb) in Cont, hFNT and As-22 cells and the results of Western blottingusing FROUNT antibody. FIG. 14 shows confocal fluoromicroscopicobservation results of eGFP-expressing cells in Cont and AS-22 cells(virus-transfer cells, upper), expression of FROUNT protein (medium) andoverlapped image thereof (lower). Compared with the Cont cells, aremarkable lowering in the FROUNT protein expression was observed in theeGFP-expressing cells (virus-transfer cells).

Subsequently, the function of FROUNT protein in cells was analyzed byusing this FROUNT antisense-transfer cells (AS-22 cells).

(2) Construction of Partly Deficient FROUNT Protein Expression Vectorand Establishment of Cell

In order to establish a cell line inhibiting the binding of endogenousFORUNT to CCR2 and analyze its phenotype, the following vector wasconstructed by forcibly expressing the C-terminal part alone of FROUNTprotein 1 carrying the binding activity to CCR2 identified by theabove-described yeast two-hybrid method and the in vitro bindingexperiment. Using as a template a plasmid vector encoding 156 C-terminalamino acids of FROUNT protein 1 isolated by using the yeast two-hybridmethod, a fragment of SEQ ID NO:38 was amplified by the PCR method withthe use of a 5′-primer: 5′GCGAATTCGCCGGATCCCGCCGCGTCGAC3′ (SEQ ID NO:57)and a 3′-primer: 5′GCGAATTCGGGGTTTTTC AGTATCTACG3′ (SEQ ID NO:58) andthen treated with EcoR1. Then it was inserted in frame into theC-terminus as allowing fusion to thereby construct a plasmid vectorDN-FNT (FIG. 12, upper). Then a virus was prepared as described aboveand a cell line was infected to establish partly deficient FROUNTprotein-expressing cells (DN-hFNT cells).

(3) Analysis of FROUNT Protein Function Using the Above Gene-TransferCells (Cont, hFNT, AS-22 and DN-hFNT Cells)

Reactivities in the FROUNT protein antisense gene-transfer cells (AS-22cells) and the partly deficient FROUNT protein-expressing cells (DN-hFNTcells) were compared and examined in the following 3 experimental lines(3-1 to 3-3) with the use of cells into which the control vector(pEGFPMY) and the FROUNT sense gene had been similarly transferred.

(3-1) Chemotactic Activity Upon Chemokine Stimulation

Participation of FROUNT protein in the cell chemotaxis induced bychemokine stimulation was examined in accordance with the method of Fallet al. (J. Immunol. Methods., 33,239-247 (1980)). Chemokines dissolvedin medium A (RPMI/0.5% BSA) at various concentrations (26 μl) weresupplied into the lower chamber of a 96-well micro-chemotaxis chamber(Neuroprobe, 5 μm), while the gene-transfer THP-1 cells dissolved inmedium A at a concentration of 10⁷ cells/ml (28 μl) were supplied intothe upper chamber and incubated at 37° C. for 30 min. Then 20 μl of 4%paraformaldehyde was added to the lower chamber to immobilize themigrating cells at 4° C. When the cells were counted with afluorocytometer, it was observed that the chemotactic activities of theAS-22 and DN-hFNT cells toward CCL2 and CCL5 were remarkably loweredcompared with the Cont cells and the hFNT cells. However, no differencein chemotactic activity toward CXCL12 was observed among the Cont, hFNT,AS-22 and DN-hFNT cells (FIG. 15). FIG. 15 shows the number of migratingcells (the longitudinal axis) in the cells (Cont, hFNT, AS-22 andDN-hFNT) when stimulated with 33 ng/ml of CCL2 or CXCL12 and FIG. 15(b)shows the number of migrating cells (the longitudinal axis) inrespective cells when stimulated with CCL2 at various concentrations.

(3-2) Increase in Intracellular Calcium Concentration (Ca²⁺) in Responseto Chemokine Stimulation

It is known that a chemokine reacts with a chemokine receptor and thuselevates intracellular calcium concentration (Ca²⁺). Thus, 10⁷gene-transfer THP-1 cells as described above were washed with PBS (GibcoBRL), suspended in 1 ml of buffer solution A (Tyroid's Salt Solution(Gibco BRL)/0.1% BSA) and incubated to a final concentration of 5 μMFruo3AM (Dojindo) at room temperature for 1 hour. Then the cells werewashed with the buffer solution A and suspended in 2 ml of the buffersolution A. The intracellular calcium concentration (Ca²⁺) was measuredby using a fluorescent spectrophotometer Fluoroscan Ascent system(Labosystems). As a result, it was confirmed that the intracellularcalcium concentration (Ca²⁺) increasing ratios of the AS-22 cells inresponse to CCL2 and CCL5 were concentration-dependently loweredcompared with those in the Cont cells and the hFNT cells (FIG. 16). FIG.16 shows the results of calcium influx (nM, the longitudinal axis)quantification into various cells stimulated with CCL2 at variousconcentrations.

(3-3) Clusterization Ability of Chemokine Receptor Using CCR2-SpecificAntibody

When a chemokine receptor reacts with a chemokine, a plural number ofreceptor molecules aggregate and form clusters followed byinternalization. The same phenomenon is observed in the reaction betweena chemokine reactor and an antibody specific thereto. To examine theparticipation of FROUNT protein 1 in this clusterization, theabove-described gene-transfer HEK293 cells, in which CCR2 was constantlyexpressed by the above-described method, were bonded to a glass plate.After washing the cells with the medium A at 4° C., a PE-labeledCCR2-specific antibody (R&D System, Inc.) was dissolved in the medium Aand incubated at 4° C. for 30 min. Then the cells were washed with themedium A and, after replacing the medium, incubated at 37° C. for 15 minfollowed by the immobilization of the cells and a treatment with aquencher. Clusterization was observed with the use of thefluoromicroscope system as described above. As a result, it wasconfirmed that the AS-22 and the DN-hFNT cells showed clearly loweringsin CCR2 clusterization ability due to the PE-labeled CCR2-specificantibody, compared with the Cont and hFNT cells (FIG. 17). FIG. 17(a)presents images of the cells stimulated with the CCR2-specific antibody,while FIG. 17(b) shows the results of the confocal microscopicquantification of the clusterization ability of each receptor stimulatedwith the CCR2- or CXCL2-specific antibody (the longitudinal axisreferring to control %).

(3-4) Inhibition of Chemokine Receptor Clusterization UsingMCP-1-Specific Antibody

To examine whether or not the clusterization of FROUNT protein 1 isinhibited by an MCP-1-speific antibody, the above-describedgene-transfer HEK293 cells, in which CCR2 had been constantly expressedby the above-described method, were bonded to a chamber slide system(Lab-Tek). Then the cells were washed with the medium A at 37° C.,replaced and incubated for 1 hour. Separately, an MCP-1 solution (100ng/ml), a liquid mixture of MCP-1 with an MCP-1-specific antibody andthe medium A alone (control) were each pre-incubated at 37° C. for 15min and then added to the cells having been incubated for 1 hour asdescribed above followed by incubation at 37° C. for additional 15minutes. Clusterization was observed with the use of a fluoromicroscopesystem. As a result, it was confirmed that clusterization of FROUNTprotein was observed in the case of adding the MCP-1 solution (see FIG.20(a)) but not in the case of adding the MCP-1-specific antibody mixture(FIG. 20(b)) and in the case of the medium A alone. It was thusconfirmed that the MCP-1-specific antibody inhibited the clusterizationof FROUNT protein in response to the chemokine stimulation. This factindicates that a FROUNT protein clusterization inhibitor can be screenedby using the gene-transfer HEK293 cells as described above.

EXAMPLE 9 Analysis of Function of FROUNT Protein 1 UsingAntisense-Transfer Mouse

To clarify the importance of FROUNT protein 1 at the individual animallevel, an antisense-transfer mouse was constructed in the followingmethod and the phenotype of the mouse was observed.

(1) Construction of Antisense-Transfer Mouse

Using the isolated mouse FROUNT gene as described above as a template, afragment was amplified by the PCR method with the use of a5′-primer:5′GCGGATCCATGGAGGAGCTCGATGGCG AGCC3′ (SEQ ID NO:59) and a3′-primer: 5′GCGGATCCTCAGGAA CCTTCCAGTGAGC3′ (SEQ ID NO:60) and treatedwith BamHI. Next, it was inserted into pEGFPMY to thereby constructantisense and sense-expression retrovirus vectors (FIG. 18). FIG. 18shows schematic models of the retrovirus vectors pEGFPMY, wherein Contstands for a control (empty) vector; mFNT stands for a mouse FROUNTprotein expression vector; AS-mFNT stands for a mouse FROUNT proteinantisense expression vector; LTR stands for a long terminal repeat; gagstands for a structural protein; eGFP stands for a green fluorescentprotein; and IRES stands for a ribosomal entry site. These vectors weretransferred into virus packaging cells BOSC23 (ATCC, CRL11554). 48 hoursafter the transformation, the cell supernatant containing therecombinant retroviruses was collected. Then bone marrow precursor cellspurified from mouse bone marrow cells with the use of an MACS system(Milteny Biotech) were infected with these retroviruses by thecentrifugation method. Cells infected with each virus vector alone wereseparated and purified to a purity of 98% or higher by using a cellsorter system with the expression of EGFP encoded by the virus vector asan indication. After irradiating recipient G57BL6 mice at lethalradiation dose (11 Gy), the virus vector-infected cells wereintravenously administered. 3 months after the transfer, 50 μl ofperipheral blood was collected from the eye ground and thereconstruction ratio of the bone marrow cells was measured with afluorocytometer. As a result, the expression of EGFP was confirmed in 70to 90% of leukocytes, which suggested that the bone marrow-origin cellshad been almost replaced by the cells infected with each virus vector.Using these mice 3 to 4 months after the transfer, the followingexperiment was carried out.

(2) Cell Migration Experiment Using Thioglycolate

In peritonitis models induced by intraperitoneally (I.P.) injectingthioglycolate into mice, a CCR2-knockout mouse shows largely reducedmacrophage infiltration after the intraperitoneal administrationcompared with a normal mouse. It is thus known the macrophageinfiltration is mediated by CCR2. Using the peritonitis models, theCCR2-mediated macrophage infiltrating ability of the FROUNTantisense-transfer mouse (AS-mFNT mouse) as described above was comparedwith those of the control mouse (Cont mouse) and the sense-transfermodel (mFNT mouse) to thereby analyze the function of FROUNT protein inindividual mice. First, thioglycolate (Difco) was dissolved in PBS at aconcentration of 4% and the obtained solution was intraperitoneallyadministered in 1.5 ml portions to the gene-transfer mice as describedabove. After 72 hours, 5 ml of PBS cooled to 4° C. was furtherintraperitoneally administered and each animal was well massaged. Thenintraperitoneal cells were collected and the cell counts were compared.As a result, it was confirmed that the AS-mFNT mouse showed a remarkabledecrease in intraperitoneal cell count compared with the Cont mouse andthe mFNT mouse. However, no abnormality in intraperitoneal cell countwas observed in these mice before the stimulation with thioglycolate.Among the intraperitoneal cells thus collected, cells expressing amacrophage-specific surface marker F4/80 or MOMA-2 were immunostainedwith a specific antibody and counted with a fluocytometer. As a result,macrophage count was obviously lowered in the As-mFNT mouse. As theresults of immunostaining with the use of mouse spleen tissue sections,it was confirmed that mouse FROUNT protein 1 was also expressed in cellsexpressing F4/80 and MOMA-2. These results suggest that, even in a mouseshowing no abnormality in the CCR2 expression, the CCR2-dependent signaltransduction system was inhibited and the infiltration ability ofmacrophages was lowered by reducing FROUNT protein, indicating theimportance of FROUNT protein 1 in an individual mouse (FIG. 19). FIG. 19shows the results of a chemotaxis experiment with the use ofthioglycolate on the intraperitoneal cells (a) and the macrophages (b)of each virus vector-transfer mouse, wherein each open bar shows a cellcount without stimulation while each solid bar shows a cell count uponthioglycolate stimulation.

EXAMPLE 10 Plasmid-Containing Liposome Preparation

(1) Construction of Plasmid

A FROUNT protein expression vector was prepared by inserting the cDNArepresented by SEQ ID NO: 19 between the EcoRI and NotI sites of apUC-SRx expression vector (FEBS 333:61-66 (1993)). In this plasmidvector, the transcription of the FROUNT protein cDNA was controlled bythe SRx promoter (Nature 342:440-443 (1989)).

(2) Production of Liposome Preparation

Tetrahydrofuran was mixed with phosphatidylserine, phosphatidylcholineand cholesterol at a weight ratio of 1:4:8:2. After distilling off thetetrahydrofuran on a rotary evaporator, the lipid mixture (10 mg) wasdeposited on the container wall. 96 μg of HMG 1 nucleic acid (highmobility group 1 nuclear protein) purified from bovine thymus was mixedwith a BBS solution (2.0 μl) of the FROUNT protein DNA plasmid (300 μg)at 20° C. for 1 hour and then the resultant mixture was added to theabove-described lipids. The obtained liposome-DNA-HMG 1 complexsuspension was mixed with a portex, ultrasonicated for 3 seconds andthen stirred for 30 minutes. Purified HVJ (Z strain) was inactivated byUV irradiation (110 erg/mm² sec) for 3 minutes immediately before using.To the liposome suspension (0.5 ml, containing 10 mg of lipids) obtainedabove, BBS was added to give a total volume of 4 ml. The obtainedmixture was incubated at 4° C. for 10 minutes and then gently stirred at37° C. for 30 minutes. Unfused liposomes were removed from theHJV-liposomes by the sucrose density gradient centrifugation method.Thus, HVJ-liposomes containing the FROUNT protein expression vector(containing 10 g/ml of FROUNT protein expression vector) was obtainedfrom the upper layer of sucrose density gradient. In the same manner, aliposome preparation containing the antisense RNA and a liposomepreparation containing DNA producing the antisense RNA can be obtained.Such a preparation is injected into a target site via an injectionneedle.

INDUSTRIAL APPLICABILITY

The protein according to the present invention, which plays a differentrole from a G protein in the intracellular signal transductionmechanisms of the G protein-coupled receptors CCR2 and CCR5, indicatesthe presence of a new mechanism participating in the intracellularsignal transduction together with the G protein. That is to say, thereis a possibility that these new mechanisms in the CCR2 system and theCCR5 system would affect the efficacy and side effects of drugs inaddition to the intracellular signal transduction mechanism having beenattracted public attention concerning the relationships among receptors,G proteins and effectors so far. Therefore, the discovery of the proteinaccording to the present invention and DNA encoding the same bringsabout the provision of novel medical targets. Namely, the presentinvention provides new approaches to the treatment, prevention,diagnosis, etc. of diseases in which signal transduction pathways(monocyte and macrophage chemotaxis, calcium mobilization, receptorclusterization, etc.) participate, as well as clarification of themechanisms thereof. In practice, it is considered that functions ofmonocytes and macrophages are affected by several factors (for example,insufficiency in a receptor, insufficiency in a G protein, etc.). Owingto the clarification of the presence of a novel protein (FROUNT protein)relating to these functions by the-present inventors, it becomespossible to diversify approaches to diseases in which monocytes andmacrophages participate.

By combining the phenomenon of the association of a FROUNT protein withthe C-terminal domain of a receptor with a labeling agent as in thepresent invention, clusterization and colocalization can be visualized.Namely, a biological phenomenon called internalization based on theinteraction between a cell receptor and an agonist can be more easilygrasped, observed and detected directly with eye. Moreover, it isexpected that various cells transformed by the procedure according tothe present invention are widely applicable and usable in various fieldsin, for example, easily detecting cytotoxic substances, detectingenvironmental pollutants, examining cytotoxicity of drugs and so on.

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1. An isolated DNA encoding the amino acid sequence represented by anyone of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36 or
 38. 2. An isolated DNA represented by any one of SEQID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35 or
 37. 3. An isolated DNA of the sequence having at least 90%identity to the DNA as claimed in claim 1 or 2 and encoding apolypeptide having a function of the FROUNT protein.
 4. A FROUNT proteinhaving an amino acid sequence represented by any one of SEQ ID NOS: 2,4, 6, 8,
 10. 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.5. A polypeptide having an amino acid sequence having at least 90%identity to the amino acid sequence as claimed in claim 4 and having thefunction of the FROUNT protein.
 6. An antisense DNA or an antisense RNAinhibiting the expression of a FROUNT protein having an amino acidsequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or
 38. 7. An antisense DNA oran antisense RNA directed against the full length or a part of the DNAas claimed in any of claims 1 to
 3. 8. An antisense RNA having the fulllength or a part of the sequence represented by SEQ ID NO:39.
 9. Anantisense RNA having at least 90% identity to the sequence of the RNA asclaimed in claim 8 and inhibiting the expression of a FROUNT protein.10. DNA for producing the RNA as claimed in claim 8 or 9 which consistsof the DNA sequence represented by SEQ ID NO:40 or the full length or apart of a sequence having at least 90% identity to this sequence.
 11. Aribozyme against an RNA corresponding to a DNA encoding the amino acidsequence represented by SEQ ID NO:2 or the DNA sequence represented bySEQ ID NO:1.
 12. A plasmid containing the DNA as claimed in any ofclaims 1 to
 3. 13. A liposome preparation containing the DNA as claimedin any of claims 1 to
 3. 14. A plasmid containing the DNA as claimed inclaim 7 or
 10. 15. A liposome preparation containing the DNA or RNA asclaimed in any of claims 7 to
 10. 16. A liposome preparation containingthe ribozyme as claimed in claim
 11. 17. An isolated antibody bindingspecifically to the polypeptide as claimed in claim 4 or
 5. 18. Acomposition for treating chronic inflammatory disease or autoimmunediseases or for treating or preventing infectious diseases whichcontains as the active ingredient the plasmid or the liposomepreparation as claimed in any of claims 14 to
 16. 19. A composition fortreating atherosclerosis, chronic glomerulonephritis or multiplesclerosis, an immunomodulator or an antiallergic agent which contains asthe active ingredient the plasmid or the liposome preparation as claimedin any of claims 14 to
 16. 20. A pharmaceutical composition whichcontains as the active ingredient the DNA as claimed in any of claims 1to
 3. 21. An immunoenhancer, a self-defensive reaction promoter or acomposition for treating or preventing infectious diseases whichcontains as the active ingredient the DNA as claimed in any of claims 1to
 3. 22. An immunoenhancer, a self-defensive reaction promoter or acomposition for treating or preventing infectious diseases whichcontains as the active ingredient the plasmid or the liposomepreparation as claimed in claim 12 or
 13. 23. A method of examining thepresence or absence of an abnormality in the CCL2-CCR2 pathway or theCCL3, 4 or 5-CCR5 pathway characterized by comprising comparing the fulllength or a part of the DNA sequence as claimed in any of claims 1 to 3with a DNA sequence collected from a specimen and thus judging whetheror not the DNA collected from the specimen has an abnormality.
 24. Aprobe for examining the presence or absence of an abnormality in theCCL2-CCR2 pathway or the CCL3, 4 or 5-CCR5 pathway which consists of thefull length or a part of a sequence complementary to the DNA as claimedin any of claims 1 to
 3. 25. (cancelled)
 26. A method of identifying aninhibitor of an agonist to receptor(s) CCR2 and/or CCR5 characterized bycomprising forcibly expressing a marker-labeled FROUNT protein in a cellhaving the receptor(s) CCR2 and/or CCR5 or expressing the same, treatingthe cell with an agonist to CCR2 and/or CCR5 and a candidate for theagonist inhibitor, observing whether or not the clusterization of thereceptor(s) is induced, and thus judging whether or not the candidatehas an inhibitory effect on the agonist.
 27. A method of identifying anagonist inhibitor by using a chimeric receptor cell characterized bycomprising preparing a cell having a labeled FROUNT protein and achimeric receptor by forcibly expressing a chimeric receptor, which isobtained by integrating a DNA sequence encoding the full length or apart of a FROUNT protein-association sequence in the intracellularC-terminal domain of receptor(s) CCR2 and/or CCR5 into the intracellularC-terminal domain of the DNA sequence of a desired receptor, in a cellappropriate for the desired receptor and then forcibly expressing amarker-labeled FROUNT protein in the cell, treating the chimericreceptor cell with an agonist to the receptor and a candidate for anagonist inhibitor, then observing whether or not the clusterization ofthe receptor is induced and thus judging whether or not the candidatehas an inhibitory effect on the agonist.
 28. The identification methodas claimed in claim 27 characterized in that the FROUNTprotein-association sequence in the intracellular C-terminal domain ofreceptor(s) CCR2 and/or CCR5 is the amino acid sequence represented bySEQ ID NO:41.
 29. The identification method as claimed in claim 26 or 27characterized in that the marker-labeled FROUNT protein is a FROUNTprotein fused with a visible color fluorescent protein.
 30. Theidentification method as claimed in claim 29 wherein the visiblecolorfluorescent protein is a green fluorescent protein, a redfluorescent protein, a blue fluorescent protein or a yellow fluorescentprotein.
 31. A DNA encoding the FROUNT protein fused with a visiblecolor fluorescent protein as claimed in claim 29 or
 30. 32. A plasmidcontaining the DNA sequence as claimed in claim
 31. 33. A chimericreceptor DNA obtained by integrating a DNA sequence encoding the fulllength or a part of a FROUNT protein-association sequence in theintracellular C-terminal domain of receptor CCR2 and/or CCR5 into theintracellular C-terminal domain of the DNA sequence of a desiredreceptor.
 34. The chimeric receptor as claimed in claim 33 characterizedin that the FROUNT protein-association sequence in the intracellularC-terminal domain of receptor(s) CCR2 and/or CCR5 is the amino acidsequence represented by SEQ ID NO:41.
 35. (cancelled)
 36. A cell whereina marker-labeled FROUNT protein is forcibly expressed and receptor(s)CCR2 and/or CCR5 are further expressed therein.
 37. A cell having alabeled FROUNT protein and a chimeric receptor prepared by forciblyexpressing a chimeric receptor, which is obtained by integrating a DNAsequence encoding the full length or a part of a FROUNTprotein-association sequence in the intracellular C-terminal domain ofreceptor(s) CCR2 and/or CCR5 into the intracellular C-terminal domain ofthe DNA sequence of a desired receptor, in a cell appropriate for thedesired receptor and then forcibly expressing a marker-labeled FROUNTprotein in the cell.
 38. The cell as claimed in claim 36 or 37characterized in that the FROUNT protein-association sequence in theintracellular C-terminal domain of receptor(s) CCR2 and/or CCR5 is theamino acid sequence represented by SEQ ID NO:41.
 39. The cell as claimedin claim 36 or 37 characterized in that the marker-labeled FROUNTprotein is a FROUNT protein fused with a visible color fluorescentprotein.
 40. The cell as claimed in claim 39 wherein the visible colorfluorescent protein is a green fluorescent protein, a red fluorescentprotein, a blue fluorescent protein or a yellow fluorescent protein. 41.A method of identifying an intracellular signal transduction pathwayinhibitor depending on binding of a FROUNT protein to a receptor whichcomprises using the binding activity of the FROUNT protein to thereceptor as an indication and screening a substance inhibiting thebinding activity.
 42. The method of identifying an inhibitor as claimedin claim 26 or 27 characterized in that the identification is madedepending on a color change as an indication by using a cell whereinboth of the receptor(s) CCR2 and/or CCR5 and FROUNT protein are labeledwith visible color markers being different from each other in color. 43.A cell wherein both of a receptor(s) CCR2 and/or CCR5 and FROUNT proteinwhich are labeled with visible color markers being different from eachother in color, are expressed therein.
 44. A method of judging whetheror not a specimen contains a cytotoxic substance which comprisestreating the cell as claimed in any of claim 36 to 40 and 43 with thespecimen, then treating it with an agonist to the receptor carried bythe cell and observing whether or not clusterization or colocalizationis induced.